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Block of Specific Gap Junction Channel Subtypes by 2-Aminoethoxydiphenyl Borate (2-APB)
Abstract
2-Aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor modulator, inhibits capacitive current transients measured in normal rat kidney and human embryonic kidney 293 cells, an indication of blocking gap junction channels between these cells. Here, we used the dual whole-cell patch-clamp method to study the actions of 2-APB on gap junction channels formed by selected connexins expressed in a communication-deficient neuroblastoma cell line (N2A). 2-APB dose-dependently and reversibly blocked junctional currents of connexin (Cx) 50 gap junction channels. The concentration-inhibition curve of 2-APB on the junctional current indicated an IC(50) of 3.7 microM, lower than that of most gap junction inhibitors. At a concentration of 20 microM, 2-APB also significantly blocked junctional conductance in cell pairs coupled by Cx26, Cx30, Cx36, Cx40, and Cx45 but did not appreciably affect coupling in cell pairs expressing Cx32, Cx43, and Cx46. Although concentration inhibition curves of 2-APB on Cx36 channels were similar to Cx50 (Cx36; IC(50), 3.0 microM), IC(50) values were higher for Cx43 (51.6 microM), Cx45 (18.1 microM), and Cx46 (29.4 microM). The blocking action of 2-APB did not substantially alter transjunctional voltage-dependent gating of Cx50 gap junction channels, and recordings from poorly coupled pairs of Cx50-transfected N2A cells indicated that 2-APB reduced gap junction channel open probability without changing the main state single-channel conductance. The differential efficacy of block by 2-APB of gap junction channels formed by different connexins may provide a useful tool that could be exploited in gap junction research to selectively block certain gap junction channel subtypes.
... Several small compounds have been reported as SOCE inhibitors and are available as pharmacological tools for SOCE research ( Figure S1). Borinate 2-APB, one of the best characterised compounds, is still widely used, despite its ample polypharmacology [8][9][10][11][12] (e.g. also targeting transient receptor potential (TRP) channels and IP 3 receptors) and known hydrolytic and oxidation-sensitive liabilities [13,14]. In addition, as a SOCE modulator, 2-APB exhibits a bimodal concentration effect: when applied in lower concentration (<10 μM), it potentiates SOCE, whereas applied in higher concentration (>30 μM), it blocks SOCE in various cell lines [15,16]. ...
... Whereas 9 with the azidotetrafluorophenyl moiety blocked SOCE at 10 μM as efficiently as the parent compound GSK-7975A, the slightly larger trifluoromethyldiazirinephenyl group of 5 had a stronger impact, significantly reducing SOCE inhibition about 4-fold. Regarding the western modification of the phenolic OH group of GSK-7975A with bioorthogonal reporter handles (probes 10 and 11), we found that they were apparently less well tolerated than the eastern ring modifications (9). A compound concentration of 15 μM or higher was required for 10 and 11 to obtain maximal Ca 2+ influx inhibition, compared to the parent compound. ...
... HEK293 cells overexpressing human Orai1 (hOrai1) were irradiated for 10 min with 302 nm or 365 nm UV light using a commercially available UV lamp (8 W). Tetrafluorophenyl azide crosslinkers (6,8,9) are usually photo-activated at 302 nm, whereas phenyldiazirines (5, 7, 12) can be photo-activated at longer wavelengths (365 nm). Analysis by Western blot showed that irradiation at the higher energy wavelength (302 nm) caused aggregation of hOrai1 proteins and generation of a broad band at high molecular weight of 250 kDa ( Figure S3). ...
... In combination with donepezil or amitriptyline, MFQ is currently tested in pre-clinical models for treatment of connexin-related disorders linked to Alzheimer disease (Cx36) and neuropathic pain (Cx43), respectively 16,32 . The second connexin inhibitor, 2APB, has been shown to have cardioprotective, anti-inflammatory, and anti-oxidative properties 33,34 , inhibiting a range of connexin channels (Cx26, Cx36, Cx32, Cx40 and Cx45 HCs and GJCs) among other targets 35,36 . Understanding the molecular basis of connexin inhibition by MFQ and 2APB would enable structure-based discovery and development of drugs that target these channels in a variety of connexin-related pathologies. ...
... junctional currents in N2A cells 26 . 2APB was shown to inhibit purified Cx32 HCs and Cx32 GJCmediated dye-coupling in HeLa cells 35 , but did not affect junctional conductance in Cx32-coupled N2A cells 36 . To assess the effect of these drugs on Cx32 HCs and GJCs we performed HC dye uptake assays and gap-fluorescence recovery after photobleaching (gap-FRAP) experiments ( Fig. 1b-c, e-f). ...
Gap junction intercellular communication (GJIC) between two adjacent cells involves direct exchange of cytosolic ions and small molecules via connexin gap junction channels (GJCs). Connexin GJCs have emerged as drug targets, with small molecule connexin inhibitors considered a viable therapeutic strategy in a number of diseases. The molecular mechanisms of GJC inhibition by known small molecule connexin inhibitors remain unknown, preventing the development of more potent and connexin-specific therapeutics. Here we show that two GJC inhibitors, mefloquine (MFQ) and 2-aminoethoxydiphenyl borate (2APB) bind to Cx32 and block dye permeation across Cx32 hemichannels (HCs) and GJCs. Cryo-EM analysis shows that 2APB binds to site A, close to the N-terminal gating helix of Cx32 GJC, restricting the entrance to the channel pore. In contrast, MFQ binds to a distinct site M, deeply buried within the pore. MFQ binding to this site modifies the electrostatic properties of Cx32 pore. Mutagenesis of V37, a key residue located in the site M, renders Cx32 HCs and GJCs insensitive to MFQ-mediated inhibition. Moreover, our cryo-EM analysis, mutagenesis and activity assays show that MFQ targets the M site in Cx43 GJC similarly to Cx32. Taken together, our results point to a conserved inhibitor binding site in connexin channels, opening a new route for development of specific drugs targeting connexins.
One-Sentence Summary
Inhibition of Cx32 and Cx43 channels via functionally relevant drug binding sites
... Thus, TRPC channels may not be fully active during the ChR2(H134R)-induced contraction and 2-APB may not be eliciting its actions via blockade of TRPC channels. Another off-target effect of 2-APB is the inhibition of gap junctions (Bai et al., 2006). One of the most abundantly expressed connexins in smooth muscle is Cx43 (Figueroa and Duling, 2009), which has relatively low sensitivity to 2-APB (IC 50 = 52 μM) (Bai et al., 2006). ...
... Another off-target effect of 2-APB is the inhibition of gap junctions (Bai et al., 2006). One of the most abundantly expressed connexins in smooth muscle is Cx43 (Figueroa and Duling, 2009), which has relatively low sensitivity to 2-APB (IC 50 = 52 μM) (Bai et al., 2006). Furthermore, gap junctions are involved in the electrical propagation among neighbouring cells, but blue light-induced depolarization will affect all individual VSMCs. ...
Background and purpose:
Optogenetic control of electromechanical coupling in vascular smooth muscle cells (VSMCs) is emerging as a powerful research tool with potential applications in drug discovery and therapeutics. However, the precise ionic mechanisms involved in this control remain unclear.
Experimental approach:
Cell imaging, patch-clamp electrophysiology, and muscle tension recordings were used to define these mechanisms over a wide range of light stimulations.
Key results:
Transgenic mice expressing a channelrhodopsin-2 variant (ChR2(H134R)) selectively in VSMCs were generated. Isolated VSMCs obtained from these mice demonstrated blue light-induced depolarising whole-cell currents. Fine control of artery tone was attained by varying the intensity of the light stimulus. This arterial response was sufficient to overcome the endogenous, melanopsin-mediated light-evoked arterial relaxation observed in the presence of contractile agonists. Pharmacological analyses revealed that Ca2+entry through voltage-gated Ca2+channels, and opening of plasmalemmal depolarising channels (TMEM16A and TRPM) and intracellular IP3receptors were mandatory for the ChR2(H134R)-dependent arterial response to blue light at intensities lower than ~0.1 mW/mm2. Light stimuli of greater intensity evoked a significant Ca2+influx directly through ChR2(H134R) and produced dramatic intracellular alkalinisation of VSMCs.
Conclusions and implications:
The range of light intensity that allows optical control of arterial tone, primarily by means of endogenous channels and without substantial alteration to intracellular pH, was identified. Within this range, mice expressing ChR2(H134R) in VSMCs are a powerful experimental model for achieving accurate and tuneable optical voltage-clamp of VSMCs and finely-graded control of arterial tone, offering new avenues for the discovery of vasorelaxing drugs.
... 2-APB was also found to affect store-operated Ca 2+ channels. In N2A neuroblastoma cells, 2-APB inhibits conductance of GJs composed of Cx36, Cx40 and Cx50 (IC 50 3 µM) as well as Cx26, Cx30 and Cx45 (IC 50 18 µM) ( Bai et al., 2006). It also inhibits transient receptor potential (TRP) channels TRPC1, TRPC3, TRPC5, TRPC6, TRPC7, TRPM3, TRPM7, TRPM8 and TRPP2, yet its inhibition seems to be often incomplete ( Bai, et al. 2006;Chung et al., 2004;Colton & Zhu, 2007;Hu et al., 2004;Ma et al., 2001;Maruyama et al., 1997). ...
... In N2A neuroblastoma cells, 2-APB inhibits conductance of GJs composed of Cx36, Cx40 and Cx50 (IC 50 3 µM) as well as Cx26, Cx30 and Cx45 (IC 50 18 µM) ( Bai et al., 2006). It also inhibits transient receptor potential (TRP) channels TRPC1, TRPC3, TRPC5, TRPC6, TRPC7, TRPM3, TRPM7, TRPM8 and TRPP2, yet its inhibition seems to be often incomplete ( Bai, et al. 2006;Chung et al., 2004;Colton & Zhu, 2007;Hu et al., 2004;Ma et al., 2001;Maruyama et al., 1997). Its boro-axozolidine ring seems to be an essential part of the pharmacophore for blocking Ca 2+ release ( Table 2). ...
While gap junctions support the exchange of a number of molecules between neighboring cells, connexin hemichannels provide communication between the cytosol and the extracellular environment of an individual cell. The latter equally holds true for channels composed of pannexin proteins, which display an architecture reminiscent of connexin hemichannels. In physiological conditions, gap junctions are usually open, while connexin hemichannels and, to a lesser extent, pannexin channels are typically closed, yet they can be activated by a number of pathological triggers. Several agents are available to inhibit channels built up by connexin and pannexin proteins, including alcoholic substances, glycyrrhetinic acid, anesthetics and fatty acids. These compounds not always strictly distinguish between gap junctions, connexin hemichannels and pannexin channels, and may have effects on other targets as well. An exception lies with mimetic peptides, which reproduce specific amino acid sequences in connexin or pannexin primary protein structure. In this paper, a state-of-the-art overview is provided on inhibitors of cellular channels consisting of connexins and pannexins with specific focus on their mode-of-action and therapeutic potential.
... Inhibitory Effect of Various Compounds on the Increased Uptake of SR-101 and PI by hCMEC/D3 Cells in the Absence of Extracellular Ca 21 .Table 2 summarizes the inhibition profiles of classic hemichannels/gap. 2-APB is a broad-spectrum inhibitor of Cx gap junctions (Bai et al., 2006). 196 Kaneko et al. junction inhibitors, some clinically approved drugs, and CBXrelated compounds on hemichannel opening–enhanced transport in hCMEC/D3 cells. ...
... Cilnidipine, a dual L/N-type Ca 21 channel blocker and progesterone, a neurosteroid, each significantly reduced the increase of both SR-101 and PI uptake by hCMEC/D3 cells. Among the classic hemichannel/ gap junctions inhibitors, 2-APB, an inhibitor of Cx gap junctions (Bai et al., 2006), reduced both SR-101 and PI uptake, whereas flufenamic acid, an inhibitor of Px1 (Iglesias et al., 2008) and Cx43 (Stout et al., 2002) and Brilliant Blue G, an inhibitor of Px1 (Qiu and Dahl, 2009), inhibited SR-101 uptake. Among clinically approved drugs that have been reported to have neuroprotective effects in humans or animal models, fluvastatin, citostasol, and FK506 inhibited only SR- 101 uptake, and pravastatin and edaravone had no significant effect. ...
Dysregulation of blood-brain barrier (BBB) transport function is considered to exacerbate neuronal damage in acute ischemic stroke. The purpose of this study was to clarify the characteristics of pannexin (Px) and/or connexin (Cx) hemichannel(s)-mediated transport of organic anions and cations in human BBB endothelial cell line hCMEC/D3 and to identify inhibitors of hemichannel opening in hCMEC/D3 cells in the absence of extracellular Ca(2+), a condition mimicking acute ischemic stroke. In the absence of extracellular Ca(2+), the cells showed increased uptake and efflux transport of organic ionic fluorescent dyes. Classical hemichannel inhibitors markedly inhibited the enhanced uptake and efflux. Quantitative targeted absolute proteomics confirmed Px1 and Cx43 protein expression in plasma membrane of hCMEC/D3 cells. Knock-down of Px1 and Cx43 with siRNAs significantly inhibited the enhanced uptake and efflux of organic anionic and cationic fluorescent dyes. Clinically used cilnidipine and progesterone, which have neuroprotective effects in animal ischemia models, were identified as inhibitors of hemichannel opening. These findings suggest that altered transport dynamics at the human BBB in the absence of extracellular Ca(2+) is at least partly due to opening of Px1 and Cx43 hemichannels. Therefore, we speculate that Px1 and Cx43 may be potential drug targets to ameliorate BBB transport dysregulation during acute ischemia.
The American Society for Pharmacology and Experimental Therapeutics.
... For example, 2-APB also blocks gap junctions with IC 50 concentrations depending on the isotype (IC 50 of ~50 μM for Cx43 while ~3 μM for the neuronal Cx36). 64 Here, we found 2-APB to inhibit Cx43-based hemichannels with an IC 50 of ~20 μM (Figure 7B), that is, 2.5-fold lower than for gap junctions. Inspired by the recent identification of a selfbinding sequence in IP 3 R3 ( Figure 1A), we conceived and characterized IP3RPEP6 consisting of an IP 3 R2-based sequence that competitively interacts with IP 3 binding to the receptor site. ...
... For example, 2-APB also blocks gap junctions with IC 50 concentrations depending on the isotype (IC 50 of ~50 μM for Cx43 while ~3 μM for the neuronal Cx36). 64 Here, we found 2-APB to inhibit Cx43-based hemichannels with an IC 50 of ~20 μM (Figure 7B), that is, 2.5-fold lower than for gap junctions. Inspired by the recent identification of a selfbinding sequence in IP 3 R3 ( Figure 1A), we conceived and characterized IP3RPEP6 consisting of an IP 3 R2-based sequence that competitively interacts with IP 3 binding to the receptor site. ...
Aim
Inositol 1,4,5‐trisphosphate receptors (IP 3 Rs) are intracellular Ca ²⁺ ‐release channels with crucial roles in cell function. Current IP 3 R inhibitors suffer from off‐target effects and poor selectivity towards the three distinct IP 3 R subtypes. We developed a novel peptide inhibitor of IP 3 Rs and determined its effect on connexin‐43 (Cx43) hemichannels, which are co‐activated by IP 3 R stimulation.
Methods
IP3RPEP6 was developed by in silico molecular docking studies and characterized by on‐nucleus patch‐clamp experiments of IP 3 R2 channels and carbachol‐induced IP 3 ‐mediated Ca ²⁺ responses in IP 3 R1, 2 or 3 expressing cells, triple IP 3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch‐clamp and ATP‐release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP 3 Rs were verified by co‐immunoprecipitation and affinity pull‐down assays.
Results
IP3RPEP6 concentration‐dependently reduced the open probability of IP 3 R2 channels and competitively inhibited IP 3 Rs in an IC 50 order of IP 3 R2 (~3.9 μM) < IP 3 R3 (~4.3 μM) < IP 3 R1 (~9.0 μM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co‐immunoprecipitated with IP 3 R2 but not with IP 3 R1; interaction with IP 3 R3 varied between cell types. The IC 50 of IP3RPEP6 inhibition of carbachol‐induced Ca ²⁺ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19‐inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP 3 ‐Ca ²⁺ responses and strongly increased the EC 50 of these responses. Finally, we identified palmitoyl‐8G‐IP3RPEP6 as a membrane‐permeable IP3RPEP6 version allowing extracellular application of the IP 3 R‐inhibiting peptide.
Conclusion
IP3RPEP6 inhibits IP 3 R2/R3 at concentrations that have limited effects on IP 3 R1. IP 3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration‐dependence of IP 3 ‐triggered Ca ²⁺ responses.
... Minute changes in the environment of the NTH (such as binding of lipids or other small molecules within the NT pocket) may lead to rearrangement of the NTH in an iris-like fashion, as seen in the Cx32 HC structure, favoring the closure of the pore at its cytosolic side. It is known that the Cx32 channel function can be modulated by hydrophobic small molecules, such as 2-aminoethoxydiphenyl borate (55,56), oleamide (57), or anandamide (28, 30, 32-34, 57, 58). It remains to be determined whether the NTH arrangement observed in the Cx32 HCs can be adopted by other connexin channels. ...
In myelinating Schwann cells, connection between myelin layers is mediated by gap junction channels (GJCs) formed by docked connexin 32 (Cx32) hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a degenerative neuropathy without a cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high-resolution cryo–electron cryo-myography (cryo-EM) structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: In the W3S and R22G mutant HCs, the amino-terminal gating helix partially occludes the pore, consistent with a diminished HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X.
... The synthetic agonist 2-aminoethoxydiphenyl borate (2-APB) robustly activates rodent but not human TRPV2 channels (Hu et al., 2004;Juvin et al., 2007), and even rodent TRPV2 channels have very low apparent affinity for 2-APB, with an EC 50 > 1 mM (Gao et al., 2016a;Liu and Qin, 2016) that is close to the solubility limit of the compound. Importantly, 2-APB also targets other channels, including TRP channels (Hu et al., 2004;Xu et al., 2005;Togashi et al., 2008;Kovacs et al., 2012), STIM-Orai channels (Bootman et al., 2002), and gap-junctions (Bai et al., 2006). Cannabidiol (CBD), a non-psychotropic compound from the cannabis plant that has received much attention recently for its potential use in treating a variety of disorders (Pauli et al., 2020), has also been found to activate rodent TRPV2 channels with much higher apparent affinity than 2-APB (Qin et al., 2008). ...
The cation-permeable TRPV2 channel is important for cardiac and immune cell function. Cannabidiol (CBD), a non-psychoactive cannabinoid of clinical relevance, is one of the few molecules known to activate TRPV2. Using the patch-clamp technique, we discover that CBD can sensitize current responses of the rat TRPV2 channel to the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB) by over two orders of magnitude, without sensitizing channels to activation by moderate (40°C) heat. Using cryo-EM, we uncover a new small-molecule binding site in the pore domain of rTRPV2 in addition to a nearby CBD site that had already been reported. The TRPV1 and TRPV3 channels are also activated by 2-APB and CBD and share multiple conserved features with TRPV2, but we find that strong sensitization by CBD is only observed in TRPV3, while sensitization for TRPV1 is much weaker. Mutations at non-conserved positions between rTRPV2 and rTRPV1 in either the pore domain or the CBD sites failed to confer strong sensitization by CBD in mutant rTRPV1 channels. Together, our results indicate that CBD-dependent sensitization of rTRPV2 channels engages multiple channel regions, and that the difference in sensitization strength between rTRPV2 and rTRPV1 channels does not originate from amino acid sequence differences at the CBD binding site or the pore domain. The remarkably robust effect of CBD on TRPV2 and TRPV3 channels offers a promising new tool to both understand and overcome one of the major roadblocks in the study of these channels - their resilience to activation.
... Minute changes in the environment of the NTH (such as binding of lipids or other small molecules within the NT pocket) may lead to rearrangement of the NTH in an iris-like fashion, as seen in the Cx32 HC structure, favoring the closure of the pore at its cytosolic side. It is known that the Cx32 channel function can be modulated by hydrophobic small molecules, such as 2-APB (48,49), oleamide (50) or anandamide (28, 30, 32-34, 50, 51). The possibility of manipulating the channel via alterations of the NT-pocket opens exciting opportunities for future structure-function investigations and drug screening for the treatment of CMT1X and other connexinopathies. ...
In myelinating Schwann cells, communication between myelin layers is mediated by gap junction channels (GJC) formed by docked connexin 32 hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot–Marie–Tooth disease (CMT1X), a degenerative neuropathy with no cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high resolution cryo-EM structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: in the W3S and R22G mutant HCs, the N-terminal helix partially occludes the pore, consistent with an impaired HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X.
One-Sentence Summary
Connexin 32 channel structures reveal a gating helix defect in CMT1X disease-associated mutant hemichannels
... ;https://doi.org/10.1101https://doi.org/10. /2023 STIM-Orai channels (Bootman et al., 2002) and gap-junctions (Bai et al., 2006). Cannabidiol (CBD), a non-psychotropic compound from the cannabis plant that has received much attention recently for its potential use in treating a variety of disorders (Pauli et al., 2020) has also been found to activate rodent TRPV2 channels with much higher apparent affinity than 2-APB (Qin et al., 2008). ...
The cation-permeable TRPV2 channel is essential for cardiac and immune cells. Cannabidiol (CBD), a non-psychoactive cannabinoid of clinical relevance, is one of the few molecules known to activate TRPV2. Using the patch-clamp technique we discover that CBD can sensitize current responses of the rat TRPV2 channel to the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB) by over two orders of magnitude, without sensitizing channels to activation by moderate (40 °C) heat. Using cryo-EM we uncover a new small-molecule binding site in the pore domain of rTRPV2 that can be occupied by CBD in addition to a nearby CBD site that had already been reported. The TRPV1 and TRPV3 channels share >40% sequence identity with TRPV2 are also activated by 2-APB and CBD, but we only find a strong sensitizing effect of CBD on the response of mouse TRPV3 to 2-APB. Mutations at non-conserved positions between rTRPV2 and rTRPV1 in either the pore domain or the CBD sites failed to confer strong sensitization by CBD in mutant rTRPV1 channels. Together, our results indicate that CBD-dependent sensitization of TRPV2 channels engages multiple channel regions and possibly involves more than one CBD and 2-APB sites. The remarkably robust effect of CBD on TRPV2 and TRPV3 channels offers a promising new tool to both understand and overcome one of the major roadblocks in the study of these channels – their resilience to activation.
... Because astrocytic Ca 2+ elevation during spreading depolarization induced by KCl (non-ischemic conditions) is insensitive to 2-APB [58], alternative directions for the therapeutic effect of this molecule should be taken. Interestingly, 2-APB is also a recog-nized gap junction blocker with high affinity for the neuronal gap junction Connexin-36 (Cx36) [59], which has been involved in the generation of spreading depolarization [60,61]. In photothrombotic models, the nonspecific blocker of Cx36, mefloquine, reduced neuronal death; however, this compound did not show any neuroprotective effect on Cx36-deficient mice [62], which are naturally neuroprotected from brain ischemia and show a lower incidence of spreading depolarizations [60,62]. ...
Brain stroke is a highly prevalent pathology and a main cause of disability among older adults. If not promptly treated with recanalization therapies, primary and secondary mechanisms of injury contribute to an increase in the lesion, enhancing neurological deficits. Targeting excitotoxicity and oxidative stress are very promising approaches, but only a few compounds have reached the clinic with relatively good positive outcomes. The exploration of novel targets might overcome the lack of clinical translation of previous efficient preclinical neuroprotective treatments. In this study, we examined the neuroprotective properties of 2-aminoethoxydiphenyl borate (2-APB), a molecule that interferes with intracellular calcium dynamics by the antagonization of several channels and receptors. In a permanent model of cerebral ischemia, we showed that 2-APB reduces the extent of the damage and preserves the functionality of the cortical territory, as evaluated by somatosensory evoked potentials (SSEPs). While in this permanent ischemia model, the neuroprotective effect exerted by the antioxidant scavenger cholesteronitrone F2 was associated with a reduction in reactive oxygen species (ROS) and better neuronal survival in the penumbra, 2-APB did not modify the inflammatory response or decrease the content of ROS and was mostly associated with a shortening of peri-infarct depolarizations, which translated into better cerebral blood perfusion in the penumbra. Our study highlights the potential of 2-APB to target spreading depolarization events and their associated inverse hemodynamic changes, which mainly contribute to extension of the area of lesion in cerebrovascular pathologies.
... 2-APB also inhibits TRPM7 channels and connexin proteins from GAP junctions. Nevertheless, it seems unlikely that cardiopulmonary actions observed here are related to these targets because neither TRPM7 nor 2-APBsensitive connexins are upregulated in the pulmonary vasculature under chronic hypoxia, and the concentrations needed to inhibit the former are excessively high and related to acidification of intracellular milieu rather than direct action on the channel (Bai et al., 2006;Billaud et al., 2011;Chokshi et al., 2012). There is also evidence that 2-APB can act as a reactive oxygen species (ROS) scavenger, and in doing so, it protects cardiomyocytes, ovary, and testis tissue against ischemia/reperfusion damage (Taskin et al., 2014;Sari et al., 2015;Morihara et al., 2017). ...
Calcium signaling is key for the contraction, differentiation, and proliferation of pulmonary arterial smooth muscle cells. Furthermore, calcium influx through store-operated channels (SOCs) is particularly important in the vasoconstrictor response to hypoxia. Previously, we found a decrease in pulmonary hypertension and remodeling in normoxic newborn lambs partially gestated under chronic hypoxia, when treated with 2-aminoethyldiphenyl borinate (2-APB), a non-specific SOC blocker. However, the effects of 2-APB are unknown in neonates completely gestated, born, and raised under environmental hypoxia. Accordingly, we studied the effects of 2-APB-treatment on the cardiopulmonary variables in lambs under chronic hypobaric hypoxia. Experiments were done in nine newborn lambs gestated, born, and raised in high altitude (3,600 m): five animals were treated with 2-APB [intravenous (i.v.) 10 mg kg–1] for 10 days, while other four animals received vehicle. During the treatment, cardiopulmonary variables were measured daily, and these were also evaluated during an acute episode of superimposed hypoxia, 1 day after the end of the treatment. Furthermore, pulmonary vascular remodeling was assessed by histological analysis 2 days after the end of the treatment. Basal cardiac output and mean systemic arterial pressure (SAP) and resistance from 2-APB- and vehicle-treated lambs did not differ along with the treatment. Mean pulmonary arterial pressure (mPAP) decreased after the first day of 2-APB treatment and remained lower than the vehicle-treated group until the third day, and during the fifth, sixth, and ninth day of treatment. The net mPAP increase in response to acute hypoxia did not change, but the pressure area under the curve (AUC) during hypoxia was slightly lower in 2-APB-treated lambs than in vehicle-treated lambs. Moreover, the 2-APB treatment decreased the pulmonary arterial wall thickness and the α-actin immunoreactivity and increased the luminal area with no changes in the vascular density. Our findings show that 2-APB treatment partially reduced the contractile hypoxic response and reverted the pulmonary vascular remodeling, but this is not enough to normalize the pulmonary hemodynamics in chronically hypoxic newborn lambs.
... is a lipophilic compound commonly used to study ion channels. Originally used as a blocker of inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) receptors, it was later found to be able to block potassium [111], gap junction connexin [112], and various TRP channels [113], as well as store-operated calcium entry (SOCE) [114]. Specifically, within the TRPM family, 2-APB has been found to inhibit TRPM2 [115], TRPM3 [116], and TRPM7 at micromolar concentrations [72]. ...
TRPM7 is a non-selective divalent cation channel with an alpha-kinase domain. Corresponding with its broad expression, TRPM7 has a role in a wide range of cell functions, including proliferation, migration, and survival. Growing evidence shows that TRPM7 is also aberrantly expressed in various cancers, including brain cancers. Because ion channels have widespread tissue distribution and result in extensive physiological consequences when dysfunctional, these proteins can be compelling drug targets. In fact, ion channels comprise the third-largest drug target type, following enzymes and receptors. Literature has shown that suppression of TRPM7 results in inhibition of migration, invasion, and proliferation in several human brain tumours. Therefore, TRPM7 presents a potential target for therapeutic brain tumour interventions. This article reviews current literature on TRPM7 as a potential drug target in the context of brain tumours and provides an overview of various selective and non-selective modulators of the channel relevant to pharmacology, oncology, and ion channel function.
... Some isoform selectivity for inhibition of HCs by 2-aminoethoxydiphenyl borate has been reported [233], but this compound also affects GJCs and proteins such as IP 3 receptors, Ca 2+ , and TRP channels [5]. Regarding its mechanism of action on HCs, 2-aminoethoxydiphenyl borate reduces their open probability without affecting single-channel conductance [223]. ...
Connexins are membrane proteins involved directly in cell-to-cell communication through the formation of gap-junctional channels. These channels result from the head-to-head docking of two hemichannels, one from each of two adjacent cells. Undocked hemichannels are also present at the plasma membrane where they mediate the efflux of molecules that participate in autocrine and paracrine signaling, but abnormal increase in hemichannel activity can lead to cell damage in disorders such as cardiac infarct, stroke, deafness, cataracts, and skin diseases. For this reason, connexin hemichannels have emerged as a valid therapeutic target. Know small molecule hemichannel inhibitors are not ideal leads for the development of better drugs for clinical use because they are not specific and/or have toxic effects. Newer inhibitors are more selective and include connexin mimetic peptides, anti-connexin antibodies and drugs that reduce connexin expression such as antisense oligonucleotides. Re-purposed drugs and their derivatives are also promising because of the significant experience with their clinical use. Among these, aminoglycoside antibiotics have been identified as inhibitors of connexin hemichannels that do not inhibit gap-junctional channels. In this review, we discuss connexin hemichannels and their inhibitors, with a focus on aminoglycoside antibiotics and derivatives of kanamycin A that inhibit connexin hemichannels, but do not have antibiotic effect.
... Several chemical inhibitors [23][24][25][26][27] as well as potentiators of SOCE [28][29][30] have been reported. However, 2-aminoethyl diphenylborinate (2-APB, Figure 1) remains the best characterized compound used to modulate store-operated CRAC channels, despite of its effects on other known targets [31][32][33][34][35][36][37][38][39][40][41][42][43][44]. Although originally described as an inhibitor of IP3 receptors [45,46], it was later shown that inhibition of SOCE and ICRAC by 2-APB is independent of its action on IP3 receptors [36,[47][48][49][50][51]. ...
Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca2+ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP3) receptors) trigger a refilling process known as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca2+ sensing stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly selective Ca2+ channels that facilitate a measurable Ca2+ influx into the cytosol followed by successive refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune, and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of 2-APB, introducing halogen and other small substituents systematically on each position of one of the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE.
... Instead of altering single channel conductance, 2-APB inhibits gap junctions by rapidly reducing the number of channels remaining in the open state. Potency of 2-APB inhibition varies depending on specific connexin subunits (Bai et al., 2006). Roles of subunits Cx37 and Cx40 have been studied extensively in platelets using specific gap junction inhibitors and genetic knockout mice. ...
Platelets release a variety of extracellular vesicles, including platelet-derived microparticles that expose phosphatidylserine on their surface. This thesis aims to resolve the mechanisms by which pro-thrombotic microparticles are released from platelets, which have not been well-characterised. Experimental methods used include flow cytometry, Western blotting, fluorescence-based microplate assays, confocal microscopy and electron microscopy. Intact cholesterol-rich lipid rafts were found to be required for the calpain-dependent release of microparticles from activated platelets. Microparticle release was prevented when cholesterol was depleted or sequestered from the platelet membrane. Membrane blebbing and scission in this process were independent of influx of hydrophilic ions or the endosomal sorting complex required for transport. It was found that microparticles were also released from platelets undergoing apoptosis, which would progress to secondary necrosis in vitro as they were not cleared. Microparticle release in apoptotic platelets was dependent on Ca2+ entry, intracellular Ca2+ mobilisation and caspase activity, but it was largely independent of calpain. During apoptosis, caspases downregulated microparticle release from platelets in response to pro-coagulant stimuli. This may limit the pro-thrombotic consequences by providing a period of reduced capacity for platelet activation. 2-Aminoethoxydiphenyl borate (2-APB), a non-specific modulator of ion channels, was found to inhibit microparticle release from both activated and apoptotic platelets. In activated platelets, the effect of 2-APB was not related to inhibition of plasma membrane ion channels or calpain activity. In apoptotic platelets, the effect of 2-APB might be associated with inhibition of Ca2+ entry, although the specific target remained unclear. Future studies in identifying the target of 2-APB might provide new insights into how microparticles are released from platelets. Moreover, 2-APB may provide a scaffold for developing a pharmacological inhibitor of microparticle release.
... unc-77(e625) animals exposed to 2-APB at both 10 and 40 μM concentrations looked indistinguishable from wild-type animals ( Although we obtained 2-APB for its K + channel activator properties, this drug has a variety of additional pharmacological actions as can be seen in Fig. 3. It inhibits inositol trisphosphate receptors (IP3R), transient receptor potential channels, store-operated calcium channels, and gap junctions made of connexin26 or connexin32 (Bootman et al., 2002;Peppiatt et al., 2003;Bai et al., 2006). Because 2-APB was the only K + channel activator to restore the performance of unc-77(e625) animals to wildtype levels, we reasoned that 2-APB might act instead on calcium channels or other types of channel to rescue locomotion. ...
The Na leak-current channel (NALCN) regulates the resting membrane potential in excitable cells, thus determining the likelihood of depolarization in response to incoming signals. Gain-of-function (gf) mutations in this channel are associated with severe dystonic movement disorders in man. Currently, there are no known pharmacological antagonists or selective modulators of this important channel. A gain-of-function mutation in NALCN of C. elegans [known as unc-77(e625)] causes uncoordinated, hyperactive locomotion. We hypothesized that this hyperactive phenotype can be rescued with pharmacological modulators. Here, we summarize the results of targeted drug screening aimed at identification of drugs that corrected locomotion deficits in unc-77(e625) animals. To assay hyperactive locomotion, animals were acutely removed from food and characteristic foraging movements were quantified. Drug screening revealed that 2-aminoethoxydiphenyl borate (2-ABP), nifedipine, nimodipine, flunarizine and ethoxzolamide significantly decreased abnormal movements in unc-77(e625) animals. 2-APB also corrected egg release and coiling deficits in this strain. In addition, serotonin and dopamine both reduced hyperactive locomotion, consistent with regulatory interactions between these systems and the NALCN. 2-APB induced movement phenotypes in wild-type animals that faithfully mimicked those observed in NALCN knockout strains, which suggested that this drug may directly block the channel. Moreover, 2-APB and flunarizine showed significant structural similarities suggestive of overlap in their mode of action. Together, these studies have revealed new insights into regulation of NALCN function and led to the discovery of a potential pharmacological antagonist of the NALCN.
... SLCO2A1 is a core component of the maxi-anion channel (68), although the precise molecular composition of the maxianion channel is still unclear (69). G proteincoupled receptor activation (70) and dephosphorylation of maxi-anion channels (71) are involved in the opening mechanisms. The presumed suppression of cAMPdependent protein kinase (PKA) downstream of Gi activation by SAG could lead to dephosphorylation of maxi-anion channels. ...
Sonic hedgehog (SHH) is important for organogenesis during development. Recent studies have indicated that SHH is also involved in the proliferation and transformation of astrocytes to the reactive phenotype. However, the mechanisms underlying these are unknown. Involvement of SHH signaling in calcium (Ca) signaling has not been extensively studied. Here, we report that SHH and Smoothened agonist (SAG), an activator of the signaling receptor Smoothened (SMO) in the SHH pathway, activate Ca oscillations in cultured murine hippocampal astrocytes. The response was rapid, on a minute timescale, indicating a non-canonical pathway activity. Pertussis toxin blocked the SAG effect, indicating an involvement of a Gi coupled to SMO. Depletion of extracellular ATP by apyrase, an ATP degrading enzyme, inhibited the SAG-mediated activation of Ca oscillations. These results indicate that SAG increases extracellular ATP levels by activating ATP release from astrocytes, resulting in Ca oscillation activation. We hypothesize that SHH activates SMO-coupled Gi in astrocytes, causing ATP release and activation of Gq/11-coupled P2 receptors on the same cell or surrounding astrocytes. Transcription factor activities are often modulated by Ca patterns; therefore, SHH signaling may trigger changes in astrocytes by activating Ca oscillations. This enhancement of Ca oscillations by SHH signaling may occur in astrocytes in the brain in vivo because we also observed it in hippocampal brain slices. In summary, SHH and SAG enhance Ca oscillations in hippocampal astrocytes, Gi mediates SAG-induced Ca oscillations downstream of SMO, and ATP-permeable channels may promote the ATP release that activates Ca oscillations in astrocytes.
... 2-aminoethoxydiphenyl borate (2-APB) is a compound initially shown to trigger IP3 Ca 2+ release in cerebellar microsomes [2]. 2-APB and analogs have been demonstrated as small molecule inhibitors (SMI) to block Cx hemichannels [3,4]. Small molecule inhibitors (SMI) that block gap junctions in liver, namely 2-APB, were shown to block communication at the hepatic gap junction and deter drug induced liver toxicity. ...
Connexins are the transmembrane pore forming proteins that participate in gap junctions; connections between cells that certain nutrients and other molecules can pass through. There are several kinds of connexins (Cx) named based on their weight in kilodaltons. 2-aminoethoxydiphenyl borate (2-APB) is a small molecule inhibitor (SMI) of Cx26 and Cx32. Knock out of Cx32 and also blockage of Cx32 by 2-aminoethoxydiphenyl (2-APB) has been suspected to be beneficial in not only, drug induced liver toxicity, but also in blocking the propagation of an inflammatory signal. If the binding site of 2-APB can be determined, virtual screening for additional, perhaps more specific Cx26 and Cx32 blocking SMI can be carried out. Our modeling suggests that 2-APB binds to similar sites inside the pores of Cx26 and Cx32. Here 2-APB interacts with the conserved ILE82 and THR86. These residues hold the same numbering in both CX26 and CX32. This suggests these residues have a high level of conservation and importance Further virtual screening results imply molecules with similar activity on Cx26 and Cx32 as 2-APB can be found.
Background
2-APB has been shown to block Cx26 and Cx32.
Results
Docking of 2-APB to Cx26 and CX32 finds conserved binding site in pore. Suggestive that 2-APB has conserved homologous binding site on Cx26 and Cx32.
Conclusion
Mutational studies of 2APB and ILE82 and THR86 in Cx26 and Cx32 appear warranted. Additional virtual screening could yield 2-APB analogs that act on Cx26 and Cx32.
Significance
Potential for developing gap junction blocking compounds.
... To our surprise, five of the six gap junction blockers that we tested (carbenoxolone, quinine, mefloquine, octanol, and meclofenamic acid) eliminated spontaneous PC firing ( Figure S4). Fortunately, the gap junction blocker 2-Aminoethoxydiphenyl borate (2-APB) (Bai et al., 2006;Harks et al., 2003) did not alter PC firing, and we therefore assessed the effect of 2-APB on correlations. We found that 2-APB did not affect the correlations (Figures 3E and 3F), which indicates that correlations in PC firing are not reliant on gap junction coupling. ...
Correlated neuronal activity at various timescales plays an important role in information transfer and processing. We find that in awake-behaving mice, an unexpectedly large fraction of neighboring Purkinje cells (PCs) exhibit sub-millisecond synchrony. Correlated firing usually arises from chemical or electrical synapses, but, surprisingly, neither is required to generate PC synchrony. We therefore assessed ephaptic coupling, a mechanism in which neurons communicate via extracellular electrical signals. In the neocortex, ephaptic signals from many neurons summate to entrain spiking on slow timescales, but extracellular signals from individual cells are thought to be too small to synchronize firing. Here we find that a single PC generates sufficiently large extracellular potentials to open sodium channels in nearby PC axons. Rapid synchronization is made possible because ephaptic signals generated by PCs peak during the rising phase of action potentials. These findings show that ephaptic coupling contributes to the prevalent synchronization of nearby PCs.
... Two sets of results support the notion that growth complementation by connexin expression is the result of the presence of functional HCs: 1) Growth complementation was blocked by known HC inhibitors that included divalent cations, 2-aminoethoxydiphenyl borate, octanol, and aminoglycosides ( Figure 4) [74,79]. Inhibitors of connexin-formed channels with affinities in the low-nM range are not available, but these compounds at the concentrations employed are known to inhibit HCs [80][81][82][83]. Although non-specific, divalent cations are well-known man Cx26 HCs purified from bacteria were structurally and functionally identical to those purified from insect cells and essentially all HCs reconstituted in unilamellar liposomes were functional [72,74]. ...
Activation of connexin hemichannels is involved in the pathophysiology of disorders that include deafness, stroke, and cardiac infarct. This aspect makes hemichannels an attractive therapeutic target. Unfortunately, most available inhibitors are not selective or isoform specific, which hampers their translational application. The absence of a battery of useful inhibitors is due in part to the absence of simple screening assays for the discovery of hemichannel-active drugs. Here, we present an assay that we have recently developed to assess hemichannel function. The assay is based on the expression of functional human connexins in a genetically modified bacterial strain deficient in K⁺ uptake. These modified cells do not grow in low-K⁺ medium, but functional expression of connexin hemichannels allows K⁺ uptake and growth. This cell-growth-based assay is simple, robust, and easily scalable to highthroughput multi-well platforms.
... Similarly, the compound 2-APB was recently employed as an IP 3 R blocker to investigate the involvement of IP 3 -mediated intercellular Ca 2+ wave propagation that underlies radiation resistance of glioblastoma tumors [53]. However, 2-APB has been shown to efficiently interfere with gap junctional communication at the concentration applied in this study [306]. ...
Although radiotherapy is commonly used to treat cancer, its beneficial outcome is frequently hampered by the radiation resistance of tumor cells and adverse reactions in normal tissues. Mechanisms of cell-to-cell communication and how intercellular signals are translated into cellular responses, have become topics of intense investigation, particularly within the field of radiobiology. A substantial amount of evidence is available demonstrating that both gap junctional and paracrine communication pathways can propagate radiation-induced biological effects at the intercellular level, commonly referred to as radiation-induced bystander effects (RIBE). Multiple molecular signaling mechanisms involving oxidative stress, kinases, inflammatory molecules, and Ca(2+) are postulated to contribute to RIBE. Ca(2+) is a highly versatile and ubiquitous second messenger that regulates diverse cellular processes via the interaction with various signaling cascades. It furthermore provides a fast system for the dissemination of information at the intercellular level. Channels formed by transmembrane connexin (Cx) proteins, i.e. hemichannels and gap junction channels, can mediate the cell-to-cell propagation of increases in intracellular Ca(2+) by ministering paracrine and direct cell-cell communication, respectively. We here review current knowledge on radiation-induced signaling mechanisms in irradiated and bystander cells, particularly focusing on the contribution of oxidative stress, Ca(2+) and Cx channels. By illustrating the tight interplay between these different partners, we provide a conceptual framework for intercellular Ca(2+) signaling as a key player in modulating the RIBE and the overall response to radiation.
... Moreover, they are responsible for the intraretinal transmission of light information from ipRGCs to amacrine cells (Reifler et al., 2015). Lastly, there are reports indicating that 2-APB, used in our previous study for the inhibition of melanopsin-mediated phototransmission (Orlowska-Feuer et al., 2016), may also block gap junctions (Bai et al., 2006;Pan et al., 2007;Sekaran et al., 2007;Weng et al., 2009;Bramley et al., 2011). It is also important to note that GJs were previously shown to be involved in the expression of infra-slow oscillations in thalamocortical neurons in vitro (Lo¨rincz et al., 2009). ...
A subpopulation of olivary pretectal nucleus (OPN) neurons fire action potentials in a rhythmic manner with an eruption of activity occurring approximately every two minutes. These infra-slow oscillations depend critically on functional retinal input and are subject to modulation by light. Interestingly, the activity of photoreceptors is necessary for the emergence of the rhythm and while classic photoreceptors (rods and cones) are necessary in darkness and dim light, melanopsin photoreceptors are indispensable in bright light. Using pharmacological and electrophysiological approaches in vivo, we show that also blocking retinal gap junctions (GJs), which are expressed by multitude of retinal cells, leads to the disruption of oscillatory activity in the rat OPN. Intravitreal injection of carbenoxolone (CBX) quenched oscillations in a concentration-dependent manner with 1 mM being ineffective, 5 mM showing partial and 20 mM showing complete effectiveness in disrupting oscillations. Moreover, the most effective CBX concentration depressed cone-mediated light-induced responses of oscillatory neurons suggesting that CBX is also acting on targets other than GJs. In contrast, intravitreal injection of meclofenamic acid (MFA, 20 mM) led to disruption of the rhythm but did not interfere with cone-mediated light-induced responses of oscillatory neurons, implying that MFA is more specific towards GJs than CBX, as suggested before. We conclude that electrical coupling between various types of retinal cells and resultant synchronous firing of retinal ganglion cells is necessary for the generation of infra-slow oscillations in the rat OPN.
... We show that CBX, a connexin hemichannel inhibitor, indeed determined APs retraction suggesting a primary role exerted by purines in APs maintenance. To strengthen the involvement of connexins in ATP release, it is interesting to note that 2APB, primarily considered an IP3 receptor antagonist, has been reported as a gap junction blocker [63]. Then, APs retraction observed following treatment with 2APB matches with effects observed with CBX, enforcing the idea of ATP contribution in APs maintenance and the involvement of connexins in synaptic activity regulation [64]. ...
Astrocytes regulate neuronal activity and blood brain barrier through tiny plasma membrane branches or astrocytic processes (APs) making contact with synapses and brain vessels. Several transmitters released by astrocytes and exerting their action on several receptor classes expressed by astrocytes themselves influence their physiology. Here we found that APs are dynamically modulated by purines. In live imaging experiments carried out in rat hippocampal astrocytes, Gq-coupled P2Y1 receptor blockade with the selective antagonist MRS2179 (1 μM) or inhibition of its effector phospholipase C using U73122 (3 μM) produced APs retraction, while stimulation of the same receptor with the selective agonist 2MeSADP (100 μM) increased their number. Since astrocytes, among other transmitters, release ATP by several mechanisms including connexin hemichannels, we used the connexin hemichannel inhibitor carbenoxolone (100 μM) and APs retraction was observed. In our system we then measured expression or function of channels important for modulation of volume transmission and K+ buffering, aquaporin-4, and K+ inward rectifying (Kir) channels, respectively. Aquaporin-4 expression level did not change whereas, in whole-cell patch-clamp recordings performed to measure Kir current, we observed an increase in K+ current in all conditions where APs number was reduced. These data are supporting the idea of a dynamic modulation of astrocytic processes by purinergic signal, strengthening the role of purines in brain homeostasis.
... Another issue that should be raised is the fact that 2-APB used here to affect phototransduction of melanopsin by inhibiting transient receptor potential channels (TRPCs), has other targets in the retina. It was also shown to act as an inhibitor of vanilloid TRP family members (Hu et al., 2004), volume-regulated anion channels (Lemonnier et al., 2004) and gap junctions (Bai et al., 2006). Unfortunately, other blockers of TRPC channels, such as SKF96365 or lanthanides, also act on various channels (Merritt et al., 1990;Faber et al., 2006;Singh et al., 2010;for review, see Nilius & Flockerzi, 2014), thus restricting their use. ...
A subpopulation of olivary pretectal nucleus (OPN) neurons discharges action potentials in an oscillatory manner, with a period of approximately two minutes. This 'infra-slow' oscillatory activity depends on synaptic excitation originating in the retina. Signals from rod-cone photoreceptors reach the OPN via the axons of either classic retinal ganglion cells or intrinsically photosensitive retinal ganglion cells (ipRGCs), which use melanopsin for photon capturing. Although both cell types convey light information, their physiological functions differ considerably. The aim of the present study was to disentangle how rod-cone and melanopsin photoresponses contribute to generation of oscillatory activity. We used pharmacological manipulations of specific phototransduction cascades whilst recording extracellular single-unit activity in the OPN of anaesthetized rats. Our results show that under photopic conditions (bright light) ipRGCs play a major role in driving infra-slow oscillations, since blocking melanopsin phototransmission abolishes or transiently disturbs oscillatory firing of the OPN neurons. On the other hand, blocking rod-cone phototransmission does not change firing patterns in photopic conditions. However, under mesopic conditions (moderate light), when melanopsin phototransmission is absent, blocking rod-cone signalling causes disturbances or even the disappearance of oscillations implying that classic photoreceptors are of greater importance under moderate light. We provide evidence that all photoreceptors are required for the generation of oscillations in the OPN, although their roles in driving the rhythm are determined by the lighting conditions, consistent with their relative sensitivities. Our results further suggest that maintained retinal activity is crucial to observe infra-slow oscillatory activity in the OPN. This article is protected by copyright. All rights reserved.
... The V j -gating property of cell pairs expressing either Cx50 or its mutants was measured by dual whole-cell voltage-clamp technique as described earlier [24,29,30]. Briefly, the transfected cells were replated on glass coverslips and then transferred to a recording chamber on an inverted microscope (Leica DM IRB, Wetzlar, Germany) filled with extracellular fluid (ECF) at room temperature. ...
Gap junction (GJ) channels provide direct passage for ions and small molecules to be exchanged between neighbouring cells and are crucial for many physiological processes. GJ channels can be gated by transjunctional voltage (known as Vj-gating) and display a wide range of unitary channel conductance (γj), yet the domains responsible for Vj-gating and γj are not fully clear. The first extracellular domain (E1) of several connexins has been shown to line part of their GJ channel pore and play important roles in Vj-gating properties and/or ion permeation selectivity. To test roles of the E1 of Cx50 GJ channels, we generated a chimera, Cx50Cx36E1, where the E1 domain of Cx50 was replaced with that of Cx36, a connexin showing quite distinct Vj-gating and γj from those of Cx50. Detailed characterizations of the chimera and three point mutants in E1 revealed that, although the E1 domain is important in determining γj, the E1 domain of Cx36 is able to effectively function within the context of the Cx50 channel with minor changes in Vj-gating properties, indicating that sequence differences between the E1 domains in Cx36 and Cx50 cannot account for their drastic differences in Vj-gating and γj. Our homology models of the chimera and the E1 mutants revealed that electrostatic properties of the pore-lining residues and their contribution to the electric field in the pore are important factors for the rate of ion permeation of Cx50 and possibly other GJ channels.
... 2-Aminoethoxydiphenyl borate (2-APB) is a synthetic compound that was originally introduced as a membrane-permeable inhibitor of intracellular inositol 1,4,5 trisphosphate receptors (Bootman et al., 2002), and has been used extensively as a modulator of many kinds of ion transport proteins (Bilmen et al., 2002;Lemonnier et al., 2004;Bai et al., 2006;Clapham, 2007;Chokshi et al., 2012;Amcheslavsky et al., 2014;Takahashi et al., 2014). Recently, 2-APB has been identified as a universal agonist of TREK/TRAAK subfamily members. ...
2-Aminoethoxydiphenyl borate (2-APB) has been recently identified as a common agonist of TREK/TRAAK channels, a subfamily of two-pore domain K(+) (K2P) channels. TREK-2 displays much higher sensitivity to 2-APB compared with TREK-1, despite that these two channels share the highest homology among K2P members. However, the structural basis for their difference in response to 2-APB still remain unknown. Here we identified that the cytosolic Ct domain plays a dominant role in controlling the stimulatory effects of 2-APB on TREK-2 channel. The distal Ct region negatively regulates the effect of 2-APB, while the proximal Ct is sufficient to evoke the full 2-APB activation of the channel. Further mapping within the proximal Ct revealed that His368 is required for 2-APB activation, and the cooperation of the other non-conserved residues is also necessary. We also identified a secondary active site for 2-APB, which is located at the bottom of the transmembrane segment M2. Finally, we demonstrated that the key residues or domains required for 2-APB activation are not involved in the gating mechanism of the selectivity filter. In summary, we reveal a unique modulatory model of TREK-2-Ct that distinguishes it from TREK-1 in high sensitivity to 2-APB. The cooperation of the non-conserved residues within the proximal Ct of TREK-2 plays a dominant role in the 2-APB induced channel opening, whereas the distal Ct negatively regulates the process.
Copyright © 2015. Published by Elsevier Ltd.
... Recent evidence points to a critical involvement of IP 3 R-linked Ca 2+ signals for the spreading of cytochrome−c induced apoptosis in cellular systems coupled by gap junction channels [64,65]. Consistent with this tenet, we found that 2-APB, a well known blocker of IP 3 R-dependent calcium release [54] that also inhibits gap junction channels [66], reduced significantly Ca 2+ wave speed and caused the most pronounced reduction in both ∆Ca 2+ and ∆NO bystander response amplitudes ( Figure 5). ...
Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding "bystander" cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca2+-dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy.
Mutations in more than half of human connexin genes encoding gap junction subunits have been linked to inherited human diseases. Functional studies of human gap junction (GJ) channels are essential for revealing mechanistic insights on the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.
Connexin gap junction channels and hemichannels play important roles in intercellular communication and signaling. Some of connexin isoforms are associated with diseases, including hereditary neuropathies, heart disease and cancer. Although small molecule inhibitors of connexins show promise as therapeutic agents, the molecular mechanisms of connexin channel inhibition are unknown. Here, we report the cryo-EM structure of connexin-36 (Cx36) bound to an anti-malarial drug mefloquine at 2.1 Å resolution. Six drug binding sites partially occlude the pore of each connexon forming the channel. Each drug molecule in the ring makes contacts with residues in the pore-lining pocket and with the neighbouring mefloquine molecules, partially occluding the pore and modifying the pore electrostatics, ultimately reducing solute translocation through the channel. Structures of Cx36 in the presence of quinine and quinidine show a similar mode of drug binding. Molecular dynamics simulations of Cx36 bound to mefloquine show that drug binding affects the kinetics of ion passage through the pore. This previously undescribed mode of connexin channel inhibition presents an opportunity for designing subtype-specific connexin inhibitors.
One-sentence summary
Mechanism of connexin channel inhibition by small molecules
Aims:
CRISPR/Cas9 gene-edits of cardiac ryanodine receptor (RyR2) in human induced pluripotent stem cells derived-cardiomyocytes (hiPSC-CMs) provides a novel platform for introducing mutations in RyR2 Ca2+ binding residues and examining the resulting EC-coupling remodeling consequences.
Methods and results:
Ca2+-signaling phenotypes of mutations in RyR2 Ca2+ binding site residues associated with cardiac arrhythmia (RyR2-Q3925E) or not proven to cause cardiac pathology (RyR2-E3848A) were determined using ICa- and caffeine-triggered Ca2+ releases in voltage-clamped and TIRF-imaged wild type (WT) and mutant cardiomyocytes infected with SR-targeted ER-GCaMP6 probe. 1) ICa- and caffeine-triggered Fura-2 or ER-GCaMP6 signals were suppressed, even when ICa was significantly enhanced in Q3925E and E3848A mutant cardiomyocyte; 2) Spontaneous beating (Fura-2 Ca2+-transients) persisted in mutant cells without the SR-release signals; 3) While 5-20mM caffeine failed to trigger Ca2+-release in voltage-clamped mutant-cells, only ∼20% to ∼70% of intact myocytes responded respectively to caffeine; 4) 20mM caffeine-transients, however, activated slowly, were delayed, and variably suppressed by 2-APB, FCCP, or ruthenium red.
Conclusion:
Mutating RyR2 Ca2+ binding residues, irrespective of their reported pathogenesis, suppressed both ICa- and caffeine-triggered Ca2+ releases, suggesting interaction between Ca2+ and caffeine binding-sites. Enhanced transmembrane calcium influx and remodeling of EC-coupling pathways may underlie the persistence of spontaneous beating in CICR-suppressed mutant myocytes.
Genetically encoded fluorescent indicators are powerful tools for tracking cellular dynamic processes. Engineering these indicators requires balancing screening dimensions with screening throughput. Herein, we present a functional imaging-guided photoactivatable cell selection platform, Faculae (functional imaging-activated molecular evolution), for linking microscopic phenotype with the underlying genotype in a pooled mutant library. Faculae is capable of assessing tens of thousands of variants in mammalian cells simultaneously while achieving photoactivation with single-cell resolution in seconds. To demonstrate the feasibility of this approach, we applied Faculae to perform multidimensional directed evolution for far-red genetically encoded calcium indicators (FR-GECIs) with improved brightness (Nier1b) and signal-to-baseline ratio (Nier1s). We anticipate that this image-based pooled screening method will facilitate the development of a wide variety of biomolecular tools.
Gap junctions are integral membrane proteins that play a role in cell-to-cell communication. They are coded by the functional genes called connexins in chordates and innexins in invertebrates. However, recently pannexins were also found in mammalian genomes, which are homologous to insect innexins. Gap junction intercellular communication (GJIC) has different functions ranging from their role in ontogenesis to the transfer of intracellular signal molecules and minimizing the adverse effects of xenobiotics by dilution and steady-state catabolism. Perturbations of these gap junctions are known to promote cancers besides, and many tumor inducers reduce the functioning of these gap junctions. Insect gap junctions play a crucial role in the development of insects and perhaps might be one of the reasons for the success of insects on terrestrial habitats. Majority of the work on innexins was done on Drosophila melanogaster Meigen, 1830 (Diptera: Drosophilidae), and its innexins regulate size of the central nervous system, embryonic gut formation, metamorphosis, and the fertility of germ lines in the reproductive system. There are several insecticides like cyclodienes, organochlorines, phenypyrazoles, synthetic pyrethroids, avermectins, milbemycins, oxadiazines, semicarbazones, meta-diamides, isoxazolines which target ion channels in the insects, but there is no evidence that supports the possible toxic effects of insecticides on insect gap junctions. In this review, we discuss the importance of insect gap junctions and how they could be a potential target for chemical pest management.
Electrically excitable cells such as neurons transmit long-distance calcium or electrical signals to regulate their physiological functions. While the molecular underpinnings and down-stream effects of these intercellular communications in excitable cells have been well appreciated, little is known about whether and how non-excitable cancer cells spontaneously initiate and transmit long-distance intercellular signals. Here we report that non-excitable human colon and prostate cancer cells spontaneously initiate and spread intercellular calcium waves, in vitro and ex vivo. Xenograft model studies suggest that these calcium signals promote the growth rate of tumors in mice. Pharmacological studies elucidated that the inositol-trisphosphate-receptor (IP3R)-regulated calcium release from endoplasmic reticulum (ER), which is activated by the Gq-PLC-IP3R pathway, is a major cause for the initiation of spontaneous calcium transients. Further, the spatial-temporal characteristics of calcium dynamics can be tuned by the culture substrates of different mechanical stiffnesses. Our results provide evidence that calcium dynamics enables long-distance functional communication in non-excitable cancer cells and offer the potential to modulate calcium signaling for new cancer therapies.
HIV-associated neurological dysfunction is observed in more than half of the HIV-infected population, even in the current antiretroviral era. The mechanisms by which HIV mediates CNS dysfunction are not well understood but have been associated with the presence of long-lasting HIV reservoirs. In the CNS, macrophage/microglia and a small population of astrocytes harbor the virus. However, the low number of HIV-infected cells does not correlate with the high degree of damage, suggesting that mechanisms of damage amplification may be involved.
Here, we demonstrate that the survival mechanism of HIV-infected cells and the apoptosis of surrounding uninfected cells is regulated by inter-organelle interactions among the mitochondria/Golgi/endoplasmic reticulum system and the associated signaling mediated by IP3 and calcium. We identified that latently HIV-infected astrocytes had elevated intracellular levels of IP3, a master regulator second messenger, which diffuses via gap junctions into neighboring uninfected astrocytes resulting in their apoptosis. In addition, using laser capture microdissection, we confirmed that bystander apoptosis of uninfected astrocytes and the survival of HIV-infected astrocytes were dependent on mitochondrial function, intracellular calcium, and IP3 signaling. Blocking gap junction channels did not prevent an increase in IP3 or inter-organelle dysfunction in HIV-infected cells but reduced the amplification of apoptosis into uninfected neighboring cells. Our data provide a mechanistic explanation for bystander damage induced by surviving infected cells that serve as viral reservoirs and provide potential targets for interventions to reduce the devastating consequences of HIV within the brain.
Background:
Ventricular fibrillation is an electrophysiological disorder leading to cardiac arrest that can be caused using chemicals. The 2-aminoethoxydiphenyl borate (2-apb) is a poorly understood compound that modulates store operated calcium entry and gap junctions and can provoke ventricular fibrillation. Our study aimed to investigate the effect of 2-apb on the work of an isolated rat heart and coronary vessels under normoxic conditions, as well as under conditions of hypoxia/reoxygenation, that affect intracellular calcium.
Methods:
In order to accomplish this task, we used Langendorff rat heart preparation and multi-electrode registration of bioelectric activity of the heart with flexible arrays. An analysis of changes in the volume of coronary blood flow was also performed.
Results:
Arrhythmogenic effect of 2-apb on an isolated rat heart was shown: an increase in the frequency and variability of the heart rhythm, a decrease in the electrical conductivity of the myocardium, and the appearance of ventricular fibrillation. Under hypoxic conditions, the arrhythmogenic effect of 2-apb decreased and no ventricular fibrillation was observed. In addition, 2-apb had a stabilizing effect on coronary vessels and weakened the effect of reoxygenation on the electrical activity of the heart.
Conclusions:
Obtained results indicate that the effect of arrhythmogenic chemicals, for example, proarrhythmic drugs that affect the myocardial [Ca<sup>2+</sup>] in , depended on the oxygen supply to the heart. The components of the store operated calcium entry and gap junctions can become promising therapeutic targets for controlling the physiological disorders of the heart and blood vessels caused or accompanied by reoxygenation.
Gap junction channels (GJs) are oligomers of connexins forming channels linking neighbouring cells. GJs formed by different connexins show distinct unitary channel conductance (γj), transjunctional-voltage dependent gating (Vj-gating) properties, and modulation by intracellular magnesium ([Mg2+]i). The underlying molecular determinants are not fully clear. Previous experimental evidence indicates that residues in the amino terminal (NT) and initial segment of the first extracellular domain (E1) influence the γj, Vj-gating, and/or [Mg2+]i modulation in several GJs. Increasing negatively charged residues in Cx50 E1 (G46D or G46E) increased γj, while increasing positively charged residue (G46K) reduced the γj Sequence alignment of Cx50 and Cx37 in the NT and E1 domains revealed that in Cx50 G8 and V53 positions are negatively charged residues in Cx37 (E8 and E53, respectively). To evaluate these residues together, we generated a triple variant in Cx50, G8E, G46E, and V53E simultaneously to study its γj, Vj-gating properties, and modulation by [Mg2+]i Our data indicate that the triple variant and individual variants G8E, G46E, and V53E, significantly increased Cx50 GJ γj without a significant change in the Vj-gating. In addition, elevated [Mg2+]i reduced γj in Cx50 and all the variant GJs. These results and our homology structural models suggest that these NT/E1 residues are likely to be pore-lining and the variants increased the negative electrostatic potentials along the GJ pore to facilitate the γj of this cation preferring GJ channel. Our results indicate that electrostatic properties of the Cx50 GJ pore are important for the γj and the [Mg2+]i modulation.
The peculiar organization of cells of the gastrointestinal tract, in part, is due to the integrity of the anatomical architecture of the linkages or junctions between the neighboring cells. In this chapter, the structural and functional characteristics of these junctions are discussed.
Vascular smooth muscle contraction is suppressed by feedback dilation mediated by the endothelium. In skeletal muscle arterioles, this feedback can be activated by Ca²⁺ signals passing from smooth muscle through gap junctions to endothelial cells, which protrude through holes in the internal elastic lamina to make contact with vascular smooth muscle cells. Although hypothetically either Ca²⁺ or inositol trisphosphate (IP3) may provide the intercellular signal, it is generally thought that IP3 diffusion is responsible. We provide evidence that Ca²⁺ entry through L-type voltage-dependent Ca²⁺ channels (VDCCs) in vascular smooth muscle can pass to the endothelium through positions aligned with holes in the internal elastic lamina in amounts sufficient to activate endothelial cell Ca²⁺ signaling. In endothelial cells in which IP3 receptors (IP3Rs) were blocked, VDCC-driven Ca²⁺ events were transient and localized to the endothelium that protrudes through the internal elastic lamina to contact vascular smooth muscle cells. In endothelial cells in which IP3Rs were not blocked, VDCC-driven Ca²⁺ events in endothelial cells were amplified to form propagating waves. These waves activated voltage-insensitive, intermediate-conductance, Ca²⁺-activated K⁺ (IKCa) channels, thereby providing feedback that effectively suppressed vasoconstriction and enabled cycles of constriction and dilation called vasomotion. Thus, agonists that stimulate vascular smooth muscle depolarization provide Ca²⁺ to endothelial cells to activate a feedback circuit that protects tissue blood flow.
T-box 18 (TBX18) plays a crucial role in the formation and development of the head of the sinoatrial node. The objective of this study was to induce adipose-derived stem cells (ADSCs) to produce pacemaker-like cells by transfection with the TBX18 gene. A recombinant adenovirus vector carrying the human TBX18 gene was constructed to transfect ADSCs. The ADSCs transfected with TBX18 were considered the TBX18-ADSCs. The control group was the GFP-ADSCs. The transfected cells were co-cultured with neonatal rat ventricular cardiomyocytes (NRVMs). The results showed that the mRNA expression of TBX18 in TBX18-ADSCs was significantly higher than in the control group after 48 h and 7 days. After 7 days of co-culturing with NRVMs, there was no significant difference in the expression of the myocardial marker cardiac troponin I (cTnI) between the two groups. RT-qPCR and western blot analysis showed that the expression of HCN4 was higher in the TBX18-ADSCs than in the GFP-ADSCs. The If current was detected using the whole cell patch clamp technique and was blocked by the specific blocker CsCl. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) showed approximately twice the current density compared with the ADSCs. Our study indicated that the TBX18 gene induces ADSCs to differentiate into pacemaker‑like cells in the cardiac microenvironment. Although further experiments are required in order to assess safety and efficacy prior to implementation in clinical practice, this technique may provide new avenues for the clinical therapy of bradycardia.
Calcium signaling through store operated channels (SOC) is involved in hypoxic pulmonary hypertension. We determined whether a treatment with 2-aminoethyldiphenylborinate (2-APB), a compound with SOC blocker activity, reduces pulmonary hypertension and vascular remodeling. Twelve newborn lambs exposed to perinatal chronic hypoxia were studied, 6 of them received a 2-APB treatment and the other 6 received vehicle treatment, for 10 days in both cases. Throughout this period, we recorded cardiopulmonary variables and on day 11 we evaluated the response to an acute hypoxic challenge. Additionally, we assessed the vasoconstrictor and vasodilator function in isolated pulmonary arteries as well as their remodeling in lung slices. 2-APB reduced pulmonary arterial pressure at the third and tenth days, cardiac output between the fourth and eighth days, and pulmonary vascular resistance at the tenth day of treatment. The pulmonary vasoconstrictor response to acute hypoxia was reduced by the end of treatment. 2-APB also decreased maximal vasoconstrictor response to the thromboxane mimetic U46619 and endothelin-1 and increased maximal relaxation to 8-Br-cGMP. The maximal relaxation and potency to phosphodiesterase-5 and Rho-kinase inhibition with sildenafil and fasudil respectively, were also increased. Finally, 2-APB reduced the medial and adventitial layers' thickness, the expression of α-actin and the percentage of Ki67+ nuclei of small pulmonary arteries. Taken together, our results indicate that 2-APB reduces pulmonary hypertension, vasoconstrictor responses and pathological remodeling in pulmonary hypertensive lambs. We conclude that SOC targeting may be a useful strategy for the treatment of neonatal pulmonary hypertension, however, further testing of specific blockers is needed.
Renal autoregulation dynamics originating from the myogenic response (MR) and tubuloglomerular feedback (TGF) can synchronize over large regions of the kidney surface, likely through gap junction-mediated electrotonic conduction and reflecting distributed operation of autoregulation. We tested the hypotheses that inhibition of gap junctions reduces spatial synchronization of autoregulation dynamics, abrogates spatial and temporal smoothing of renal perfusion, and impairs renal autoregulation. In male Long-Evans rats, we infused the gap junction inhibitor, carbenoxolone (CBX), or the related glycyrrhizic acid (GZA) that does not block gap junctions, into the renal artery and monitored renal blood flow (RBF) and surface perfusion by laser speckle contrast imaging. Neither CBX nor GZA altered RBF or mean surface perfusion. CBX preferentially increased spatial and temporal variation in the distribution of surface perfusion; increased spatial variation in the operating frequencies of the MR and TGF; reduced phase coherence of TGF and increased its dispersion. CBX, but not GZA, impaired dynamic and steady-state autoregulation. Separately, infusion of the Rho kinase inhibitor Y-27632 paralyzed smooth muscle, grossly impaired dynamic autoregulation, and monotonically increased spatial variation of surface perfusion. These data suggest CBX inhibited gap junction communication which in turn reduced the ability of TGF to synchronize among groups of nephrons. The results indicate that impaired autoregulation resulted from degraded synchronization, rather than the reverse. We show that network behavior in the renal vasculature is necessary for effective RBF autoregulation.
Gap junction (GJ) channel mediates direct intercellular communication and is composed of two docked hemichannels (connexin oligomers). It is well documented that the docking and formation of GJs are possible only between compatible hemichannels (or connexins). The mechanisms of heterotypic docking compatibility are not fully clear. We aligned the protein sequences of docking compatible and incompatible connexins with that of Cx26. We found that two docking hydrogen bond-forming residues on the second extracellular domain (E2) of Cx26 and their equivalent residues are well conserved within docking compatible connexins, but different between docking incompatible connexins. Replacing one or both of these residues of Cx26 into the corresponding residues in the docking incompatible connexins (K168V, N176H or K168V-N176H) increased the formation of morphological and functional heterotypic GJs with Cx43 (or Cx40), indicating these two residues are important for docking incompatibility between Cx26 and these connexins. Our homology structure models predict that both hydrogen bonds and hydrophobic interactions at the E2 docking interface are important docking mechanisms in heterotypic Cx26 K168V-N176H/Cx43 GJs and likely other docking compatible connexins. Revealing the key residues and mechanisms of heterotypic docking compatibility will assist us in understanding why these putative docking residues are hotspots of disease-linked mutants.
Gap junction (GJ) channels provide low resistance passages for rapid action potential propagation in the heart. Both connexin40 (Cx40) and Cx43 are abundantly expressed in and frequently co-localized between atrial myocytes, possibly forming heterotypic GJ channels. However, conflicting results have been obtained on the functional status of heterotypic Cx40/Cx43 GJs. Here we provide experimental evidence that the docking and formation of heterotypic Cx40/Cx43 GJs can be substantially increased by designed Cx40 variants on the extracellular domains (E1 and E2). Specifically, Cx40 D55N and P193Q, substantially increased the probability to form GJ plaque-like structures at the cell-cell interfaces with Cx43 in model cells. More importantly the coupling conductance (Gj) of D55N/Cx43 and P193Q/Cx43 GJ channels are significantly increased from the Gj of Cx40/Cx43 in N2A cells. Our homology models indicate the electrostatic interactions and surface structures at the docking interface are key factors preventing Cx40 from docking to Cx43. Improving heterotypic Gj of these atrial connexins might be potentially useful in improving the coupling and synchronization of atrial myocardium.
During the first few cell division cycles in zebrafish, distinct Ca2+ transients are localized to the early embryonic cleavage furrows, where they accompany (and are required for) furrow positioning, propagation, deepening and apposition. It has previously been shown that the endoplasmic reticulum (ER) acts as the primary store for generating these Ca2+ transients via release through inositol 1,4,5-trisphosphate receptors (IP3Rs).We hypothesised that maintaining the elevated levels of intracellular Ca2+ required for deepening and apposition of the cleavage furrows in these large eggs might result in the depletion of the available ER Ca2+ store, thus the role of store-operated Ca2+ entry (SOCE) was examined. Newly fertilized, dechorionated embryos were incubated with various SOCE inhibitors, starting just prior to the onset of the first cell division cycle. The effect of these inhibitors on mitosis, furrow positioning, propagation, deepening and apposition, and the generation of the cytokinetic Ca2+ transients was determined. Treatment with 2-APB or SKF 96365 had no major effect on mitosis, furrow positioning or propagation, but inhibited furrow deepening resulting in regression of the cleavage furrow. Both of these inhibitors also blocked the furrowing Ca2+ transient, with SKF 96365 having a more profound inhibitory effect than 2-APB. In zebrafish, SOCE does not appear to be required for mitosis or the early stages of cytokinesis during the early embryonic cell division cycles, but it does appear to be essential for maintaining the elevated levels of [Ca2+]i for the extended periods that are required during furrow deepening and daughter cell apposition.
Vaccines currently in the clinical use contain adjuvants stimulating preferably Th2 type of immune response associated with the production of specific antibodies, mostly of neutralizing isotypes. This kind of immune response is effective only against some types of pathogens and has limited effect against tumors and many viruses where parallel activation of antigen-specific humoral and cell-mediated immunity is required. One of the main objectives of the current vaccine research is the development of approaches leading to the induction of antigen-specific CD8(+) T cell response including cytotoxic T lymphocyte (CTL). Induction of antigen-specific CD8(+) T cell response to exogenously delivered antigen requires their cross-presentation by antigen presenting cells, especially dendritic cells. The cross-presentation principles seem to be crucial for effective activation of CTL. In this paper, we discuss some approaches to employing heat shock proteins for induction of antigen-specific CD8(+) T cells in the context of cross-presentation and cross-priming principles.
Aims To determine the role of gap junctions (GJs) in hypoxic pulmonary vasoconstriction (HPV). Methods and results Studies were performed in rat isolated intrapulmonary arteries (IPAs) mounted on a myograph and in anaesthetized rats. Hypoxia induced a biphasic HPV response in IPAs preconstricted with prostaglandin F 2a (PGF 2a , 3 mM) or 20 mM K +. The GJ inhibitors 18b-glycyrrhetinic acid (18b-GA, 30 mM), heptanol (3.5 mM), or 2-aminoethoxydiphenyl borate (2-APB) (75 mM) had little effect on the transient Phase 1 of HPV, but abolished the sustained Phase 2 which is associated with Ca 2+ sensitization. The voltage-dependent Ca 2+ channel blocker diltiazem (10 mM) had no effect on HPV, and did not alter the inhibitory action of 18b-GA. Sustained HPV is enhanced by high glucose (15 mM) via potentiation of Ca 2+ sensitization, in the presence of high glucose 18b-GA still abolished sustained HPV. Simultaneous measurement of tension and intracellular Ca 2+ using Fura PE-3 demonstrated that whilst 18b-GA abolished tension development during sustained HPV, it did not affect the elevation of intracellular Ca 2+. Consistent with this, 18b-GA abolished hypoxia-induced phosphorylation of the Rho kinase target MYPT-1. In anaesthetized rats hypoxia caused a biphasic increase in systolic right ventricular pressure. Treatment with oral 18b-GA (25 mg/kg) abolished the sustained component of the hypoxic pressor response. Conclusion These results imply that GJs are critically involved in the signalling pathways leading to Rho kinase-dependent Ca 2+ sen-sitization during sustained HPV, but not elevation of intracellular Ca 2+ , and may explain the dependence of the former on an intact endothelium.
Since its introduction to Ca2+ signaling in 1997, 2-aminoethoxydiphenyl borate (2-APB) has been used in many studies to probe for the involvement of inositol 1,4,5-trisphosphate receptors in the generation of Ca2+ signals. Due to reports of some nonspecific actions of 2-APB, and the fact that its principal antagonistic effect is on Ca2+ entry rather than Ca2+ release, this compound may not have the utility first suggested. However, 2-APB has thrown up some interesting results, particularly with respect to store-operated Ca2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2-APB can be useful in probing certain aspects of Ca2+ signaling.—Bootman, M. D., Collins, T. J., Mackenzie, L., Roderick, H. L., Berridge, M. J., Peppiatt, C. M. 2-Aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2+ entry but an inconsistent inhibitor of InsP3-induced Ca2+ release.
Gap junctions are conductive channels that connect the interiors of coupled cells. We determined whether gap junctions propagate transcellular signals during metabolic stress and whether such signaling exacerbates cell injury. Although overexpression of the human proto-oncogene bcl2 in C6 glioma cells normally increased their resistance to injury, the relative resistance of bcl2+ cells to calcium overload, oxidative stress and metabolic inhibition was compromised when they formed gap junctions with more vulnerable cells. The likelihood of death was in direct proportion to the number and density of gap junctions with their less resistant neighbors. Thus, dying glia killed neighboring cells that would otherwise have escaped injury. This process of glial 'fratricide' may provide a basis for the secondary propagation of brain injury in cerebral ischemia.
We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.
Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction-mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18beta-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction.
Protonated aminosulfonate compounds directly inhibit connexin channel activity. This was demonstrated by pH-dependent connexin channel activity in Good's pH buffers (MES (4-morpholineethanesulfonic acid)), HEPES, and TAPS (3-({[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]amino]-1-propanesulfonic acid)) that have an aminosulfonate moiety in common and by the absence of pH-dependent channel activity in pH buffers without an aminosulfonate moiety (maleate, Tris, and bicarbonate). The pH-activity relation was shifted according to the pKa of each aminosulfonate pH buffer. At constant pH, increased aminosulfonate concentration inhibited channel activity. Taurine, a ubiquitous cytoplasmic aminosulfonic acid, had the same effect at physiological concentrations. These data raise the possibility that effects on connexin channel activity previously attributed to protonation of connexin may be mediated instead by protonation of cytoplasmic regulators, such as taurine. Modulation by aminosulfonates is specific for heteromeric connexin channels containing connexin-26; it does not occur significantly for homomeric connexin-32 channels. The identification of taurine as a cytoplasmic compound that directly interacts with and modulates connexin channel activity is likely to facilitate understanding of cellular modulation of connexin channels and lead to the development of reagents for use in structure-function studies of connexin protein.
Previous studies have shown that chemical regulation of connexin43 (Cx43) depends on the presence of the carboxyl terminal (CT) domain. A particle-receptor (or "ball-and-chain") model has been proposed to explain the mechanism of gating. We tested whether the CT region behaved as a functional domain for other members of the connexin family. The pH sensitivity of wild-type and Ct-truncated connexins was quantified by use of electrophysiological and optical techniques and the Xenopus oocyte system. The CT domain of Cx45 had no role in pH regulation, although a partial role was shown for Cx37 and Cx50. A prominent effect was observed for Cx40 and Cx43. In addition, we found that the CT domain of Cx40 that was expressed as a separate fragment rescued the pH sensitivity of the truncated Cx40 (Cx40tr), which was in agreement with a particle-receptor model. Because Cx40 and Cx43 often colocalize and possibly heteromerize, we tested the pH sensitivity of Cx40tr when coexpressed with the CT domain of Cx43 (hetero-domain interactions). We found that the CT domain of Cx43 enhanced the pH sensitivity of Cx40tr; similarly, the CT domain of Cx40 restored the pH sensitivity of the truncated Cx43. In addition, the CT domain of Cx43 granted insulin sensitivity to the otherwise insulin-insensitive Cx26 or Cx32 channels. These data show that the particle-receptor model is preserved in Cx40 and the regulatory domain of one connexin can specifically interact with a channel formed by another connexin. Hetero-domain interactions could be critical for the regulation of heteromeric channels.
The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.
Calcium influx and elevation of intracellular free calcium (Ca2+i), with subsequent activation of degenerative enzymes is hypothesized to cause cell injury and death after trauma. We examined the effects of traumatic compressive injury on (Ca2+)i dynamics in spinal cord white matter. We conducted electrophysiological studies with ryanodine and inositol (1,4,5)-triphosphate (IP3) receptor agonists and antagonists in an in vitro model of spinal cord injury (SCI). A 25-30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber (37 degrees C) and injured with a modified clip (2-g closing force) for 15 sec. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential (CAP) with glass microelectrodes. The CAP decreased to 55.2+/-6.8% of control (p < 0.05) after spinal cord injury (SCI). Chelation of Ca2+i with BAPTA-AM (a high-affinity calcium chelator) promoted significantly greater recovery of CAP amplitude (83.2+/-4.2% of control; p < 0.05) after injury. Infusion of caffeine (1 and 10 mM) exacerbated CAP amplitude decline (45.1+/-5.9% of control; p < 0.05; 44.6+/-3.1% of control; p < 0.05) postinjury. Blockade of Ca2+i release through ryanodine-sensitive receptors (RyRs) with dantrolene (10 microM) and ryanodine (50 microM), conferred significant (p < 0.05) improvement in CAP amplitude after injury. On the other hand, blockade of Ca2+i with inositol (1,4,5)-triphosphate receptor (IP3Rs) blocker 2APB (10 microM) also conferred significant improvement in CAP amplitude after injury (82.9+/-7.9%; p < 0.05). In conclusion, the injurious effects of Ca2+i in traumatic central nervous system (CNS) white matter injury appear to be mediated both by RyRs and through IP3Rs calcium-induced calcium release receptors (CICRs).
Gap junctions are clustered channels between contacting cells through which direct intercellular communication via diffusion of ions and metabolites can occur. Two hemichannels, each built up of six connexin protein subunits in the plasma membrane of adjacent cells, can dock to each other to form conduits between cells. We have recently screened mouse and human genomic data bases and have found 19 connexin (Cx) genes in the mouse genome and 20 connexin genes in the human genome. One mouse connexin gene and two human connexin genes do not appear to have orthologs in the other genome. With three exceptions, the characterized connexin genes comprise two exons whereby the complete reading frame is located on the second exon. Targeted ablation of eleven mouse connexin genes revealed basic insights into the functional diversity of the connexin gene family. In addition, the phenotypes of human genetic disorders caused by mutated connexin genes further complement our understanding of connexin functions in the human organism. In this review we compare currently identified connexin genes in both the mouse and human genome and discuss the functions of gap junctions deduced from targeted mouse mutants and human genetic disorders.
Since its introduction to Ca2+ signaling in 1997, 2-aminoethoxydiphenyl borate (2-APB) has been used in many studies to probe for the involvement of inositol 1,4,5-trisphosphate receptors in the generation of Ca2+ signals. Due to reports of some nonspecific actions of 2-APB, and the fact that its principal antagonistic effect is on Ca2+ entry rather than Ca2+ release, this compound may not have the utility first suggested. However, 2-APB has thrown up some interesting results, particularly with respect to store-operated Ca2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2-APB can be useful in probing certain aspects of Ca2+ signaling.
During development of the retina, programmed cell death helps to establish the final size and distribution of various cell classes in distinct layers of the tissue. Here we show that dying cells in the developing ganglion and inner nuclear layers are clustered spatially and that gap junction inhibitors decrease the clustering of dying cells. To confirm the role of gap junctions in cell death, we induced targeted cell death via intracellular cytochrome c (C c ) and examined the induced cells and their neighbors for apoptotic morphology or caspase-3 cleavage. These studies indicate that bystander killing extends to coupled cells.
Quantitative studies of bystander killing were performed by scrape-loading retinas with C c in the presence of rhodamine dextran (RD; to identify C c -loaded cells) and by counting pyknotic cells in cryosections. Although only 1.5% of control scrape-loaded cells (RD alone) showed apoptotic morphology, 97% of C c scrape-loaded cells were pyknotic. Moreover, bystander killing extended to neighboring cells, not labeled with RD, and was reduced significantly by the gap junction inhibitors octanol and carbenoxolone. We hypothesize that dying cells in the retina generate a gap junction-permeant apoptotic signal that mediates bystander killing. This novel finding of naturally occurring bystander cell death may have important implications in the histogenesis and pathology of the nervous system.
Astrocytes in the rat thalamus display spontaneous [Ca(2+)](i) oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca(2+)](i) oscillations using slices loaded with Fluo-4 AM (5 microM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca(2+)](i) oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca(2+)](i) increases are not due to membrane-coupled PLC activation. Spontaneous [Ca(2+)](i) increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca(2+)](i) oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca(2+) entry. Increasing [Ca(2+)](o) increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca(2+) ATPase by cyclopiazonic acid also induced [Ca(2+)](i) transients in astrocytes indicating a role for cytoplasmic Ca(2+) in the induction of spontaneous oscillations. Incubation with 20 microM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases. This study indicates that Ca(2+) has a role in triggering Ca(2+) release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca(2+)](i) oscillations.
Recently, great interest has been shown in understanding the functional roles of specific gap junction proteins (connexins) in brain, lens, retina, and elsewhere. Some progress has been made by studying knockout mice with targeted connexin deletions. For example, such studies have implicated the gap junction protein Cx36 in synchronizing rhythmic activity of neurons in several brain regions. Although knockout strategies are informative, they can be problematic, because compensatory changes sometimes occur during development. Therefore, it would be extremely useful to have pharmacological agents that block specific connexins, without major effects on other gap junctions or membrane channels. We show that mefloquine, an antimalarial drug, is one such agent. It blocked Cx36 channels, expressed in transfected N2A neuroblastoma cells, at low concentrations (IC(50) approximately 300 nM). Mefloquine also blocked channels formed by the lens gap junction protein, Cx50 (IC(50) approximately 1.1 microM). However, other gap junctions (e.g., Cx43, Cx32, and Cx26) were only affected at concentrations 10- to 100-fold higher. To further examine the utility and specificity of this compound, we characterized its effects in acute brain slices. Mefloquine, at 25 microM, blocked gap junctional coupling between interneurons in neocortical slices, with minimal nonspecific actions. At this concentration, the only major side effect was an increase in spontaneous synaptic activity. Mefloquine (25 microM) caused no significant change in evoked excitatory or inhibitory postsynaptic potentials, and intrinsic cellular properties were also mostly unaffected. Thus, mefloquine is expected to be a useful tool to study the functional roles of Cx36 and Cx50.
Major histocompatibility complex (MHC) class I molecules present peptides that are derived from endogenous proteins. These antigens can also be transferred to professional antigen-presenting cells in a process called cross-presentation, which precedes initiation of a proper T-cell response; but exactly how they do this is unclear. We tested whether peptides can be transferred directly from the cytoplasm of one cell into the cytoplasm of its neighbour through gap junctions. Here we show that peptides with a relative molecular mass of up to approximately 1,800 diffuse intercellularly through gap junctions unless a three-dimensional structure is imposed. This intercellular peptide transfer causes cytotoxic T-cell recognition of adjacent, innocent bystander cells as well as activated monocytes. Gap-junction-mediated peptide transfer is restricted to a few coupling cells owing to the high cytosolic peptidase activity. We present a mechanism of antigen acquisition for cross-presentation that couples the antigen presentation system of two adjacent cells and is lost in most tumours: gap-junction-mediated intercellular peptide coupling for presentation by bystander MHC class I molecules and transfer to professional antigen presenting cells for cross-priming.
We have investigated the ability of 5-methyltetrahydrofolate (5-MTHF) and tetrahydrobiopterin (BH4) to modulate nitric oxide (NO)-independent vascular relaxations that are mediated by the sequential spread of endothelial hyperpolarization through the wall of the rabbit iliac artery by means of myoendothelial and homocellular smooth muscle gap junctions. Relaxations and subintimal smooth muscle hyperpolarizations evoked by cyclopiazonic acid were depressed by the gap junction inhibitor 2-aminoethoxydiphenyl borate, whose effects were prevented by 5-MTHF and BH4, but not by their oxidized forms folic acid and 7,8-dihydrobiopterin. Analogously, 5-MTHF and BH4, but not folic acid or 7,8-dihydrobiopterin, attenuated the depression of subintimal hyperpolarization by a connexin-mimetic peptide targeted against Cx37 and Cx40 (37,40Gap 26) and the depression of subadventitial hyperpolarization by a peptide targeted against Cx43 (⁴³Gap 26), thus reflecting the known differential expression of Cx37 and Cx40 in the endothelium and Cx43 in the media of the rabbit iliac artery. The inhibitory effects of 2-aminoethoxydiphenyl borate and 37,40Gap 26 against subintimal hyperpolarization were prevented by catalase, which destroys H2O2. 5-MTHF and BH4 thus appear capable of modulating electrotonic signaling by means of myoendothelial and smooth muscle gap junctions by reducing oxidant stress, potentially conferring an ability to reverse the endothelial dysfunction found in disease states through mechanisms that are independent of NO.
• connexin
• folic acid
• methotrexate
• gap junction
• endothelium-derived hyperpolarizing factor
The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca²⁺) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca²⁺ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.
Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca2+]i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca2+]i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca2+]i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca2+]i were abolished by loading the preparations with the Ca2+ chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP3 induced Ca2+ release, by N-ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl−)-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP3 formation. This causes Ca2+ release from intracellular stores which activates Ca2+-dependent Cl− channels.
The rat connexin40 gap junction channel is permeable to monovalent cations including tetramethylammonium and tetraethylammonium ions. Larger tetraalkyammonium (TAA(+)) ions beginning with tetrabutylammonium (TBA(+)) reduced KCl junctional currents disproportionately. Ionic blockade by tetrapentylammonium (TPeA(+)) and tetrahexylammonium (THxA(+)) ions were concentration- and voltage-dependent and occurred only when TAA(+) ions were on the same side as net K(+) efflux across the junction, indicative of block of the ionic permeation pathway. The voltage-dependent dissociation constants (K(m)(V(j))) were lower for THxA(+) than TPeA(+), consistent with steric effects within the pore. The K(m)-V(j) relationships for TPeA(+) and THxA(+) were fit with different reaction rate models for a symmetrical (homotypic) connexin gap junction channel and were described by either a one- or two-site model that assumed each ion traversed the entire V(j) field. Bilateral addition of TPeA(+) ions confirmed a common site of interaction within the pore that possessed identical K(m)(V(j)) values for cis-trans concentrations of TPeA(+) ions as indicated by the modeled I-V relations and rapid channel block that precluded unitary current measurements. The TAA(+) block of K(+) currents and bilateral TPeA(+) interactions did not alter V(j)-gating of Cx40 gap junctions. N-octyl-tributylammonium and -triethylammonium also blocked rCx40 channels with higher affinity and faster kinetics than TBA(+) or TPeA(+), indicative of a hydrophobic site within the pore near the site of block.
Pairs of SKHep1 cells, which are derived from a highly metastatic human hepatoma, were studied using the whole cell voltage clamp technique with patch-type electrodes containing CsCl as the major ionic species. In 12 of 81 cell pairs, current flow through junctional membranes was detectable; in the remaining 69 cell pairs, junctional conductance was less than the noise limit of our recording apparatus (worst case: 10 pS). Macroscopic junctional conductance (gj) in the small percentage of pairs where it was detectable ranged from 100 to 600 pS. Unitary junctional conductance (gamma j) determined in the lowest conductance pairs or after reducing conductance with a short exposure to the uncoupling agent halothane was 25-35 pS. To study properties of gap junction channels formed of connexin32, the parental SKHep1 cell line was stably transfected with a plasmid containing cDNA that encodes connexin32, the major gap junction protein of rat liver cells. In 85 of 98 pairs of voltage clamped connexin32-transfected SKHep1 cells, macroscopic gj was greater than 1 nS; gj increased with time after dissociation (from 1.8 +/- 0.6 [mean +/- SE; n = 7] nS at 2 h after plating to 9.3 +/- 2.2 [n = 9] nS, the maximal value, at 24 h). Unitary conductance of gap junction channels between pairs of transfected SKHep1 cells was measured in low conductance pairs and after reducing gj by exposure to halothane or heptanol. Histograms of gamma j values in transfected cells, in 10 experiments where greater than 100 transitions were measurable, displayed two peaks; 120-130 pS and 25-35 pS. The smaller size corresponded to channels that were occasionally detected in the parental cells. We therefore conclude that connexin32 forms gap junctions channels of the 120-130 pS size class.
The effects of halothane and ethrane on gap junction-mediated intercellular communication and on membrane excitability were examined in cultured neonatal rat cardiac myocytes using whole-cell voltage-clamp and current-clamp techniques. Excitability was maintained at doses of both anesthetics that reversibly abolished current flow through junctional membranes. The degree of reduction of junctional conductance was a steep function of the dose of anesthetic; complete block occurred at lower aqueous concentrations of halothane than ethrane. The time course for loss of communication was rapid; 90% reduction of initial junctional conductance occurred in less than 15 seconds after exposure to 2 mM halothane or 4 mM ethrane. Recovery of junctional conductance and junctional permeability to intracellularly injected Lucifer yellow was rapid and complete on washout of the anesthetics. As junctional conductance was reduced by halothane or ethrane exposure, unitary conductance of the gap junctional channels remained constant at about 50 pS. Uncoupling by these anesthetics is thus attributable to a decrease in the number of conducting channels rather than to reduction of the channel's unitary conductance. The data are discussed with regard to the possible role of this intercellular communication pathway in the arrhythmias and alterations of conduction velocity and contractility produced by volatile anesthetics.
Glycyrrhetinic acid was shown previously to inhibit intercellular gap-junctional communication between human fibroblasts. In the present study 31 derivatives of glycyrrhetinic acid were tested for their ability to inhibit communication. Eight of the compounds inhibited communication with high potency (IC50 less than 3 microM) and showed low toxicity, properties which suggest they may be useful pharmacological probes for studies of gap-junction function.
Studies of the interaction of anaesthetics with various preparations, from whole animals to organic solvents, have been continuing since Overton and Meyer found a correlation between anaesthetic potency and solubility in olive oil. Although the physiological basis of anaesthesia is far from clear, one popular hypothesis is that anaesthetics act primarily by interfering with the normal functioning of chemical synapses. This hypothesis is supported by experiments showing that these synapses are more sensitive to both local and general anaesthetics than are axons. The effects of anaesthetics on electrical synapses (gap-junctions or nexus) have not previously been studied. These ubiquitous structures, presumably responsible for cell-to-cell communication, are found in most vertebrate and invertebrate tissues. We report here the effects of several anaesthetics on electronic coupling between nerve cells, and show that electrical synapses are less sensitive to most anaesthetics than are chemical synapses and axonal membranes.
Gap junctional conductance ( gj ) in various species is gated by voltage and intracellular pH (pHi). In amphibian embryos, gj is reduced to half by a 14 mV transjunctional voltage ( Vj ), a change that in fish embryo requires approximately 28 mV. Crayfish septate axon and pairs of dissociated rat myocytes show no voltage dependence of gj over a range of Vj greater than +/- 50 mV. In fish and amphibian blastomeres , gj is steeply decreased by decrease in pHi (n, Hill coefficient: 4.5) and the apparent pKH (7.3) is in the physiological range. In crayfish septate axon the pKH is lower (6.7) and the curve is less steep (n = 2.7). Rises in cytoplasmic Ca can also decrease gj but much higher concentrations are required (greater than 0.1 mM in fish blastomeres). Voltage and pH gates on gap junctions in amphibian embryos appear independent. In squid blastomeres pH gates exhibit some sensitivity to potential, both transjunctional and between inside and outside. A pharmacology of gap junctions is being developed: certain agents block gj directly (aldehydes, alcohols, NEM in crayfish); others block by decreasing pHi (esters that are hydrolyzed by intrinsic esterases, NEM in vertebrates, and, as in the experiments demonstrating the effect of pHi, weak acids). Certain agents block pH sensitivity without affecting voltage dependence (retinoic acid, glutaraldehyde, EEDQ), further indicating separateness of pH and voltage gates. These studies demonstrate a dynamics of gap junctional conductance and variability in gating in a series of possibly homologous membrane channels.
Antibodies to the gap junction protein connexin45 (Cx45) were obtained by immunizing rabbits with fusion protein consisting of glutathione S-transferase and 138 carboxy-terminal amino acids of mouse Cx45. As shown by immunoblotting and immunofluorescence, the affinity-purified antibodies recognized Cx45 protein in transfected human HeLa cells as well as in the kidney-derived human and hamster cell lines 293 and BHK21, respectively. In Cx45-transfected HeLa cells, this protein is phosphorylated as demonstrated by immunoprecipitation after metabolic labeling. The phosphate label could be removed by treatment with alkaline phosphatase. A weak phosphorylation of Cx45 protein was also detected in the cell lines 293 and BHK21. Treatment with dibutyryl cyclic adenosine or guanosine monophosphate (cAMP, cGMP) did not alter the level of Cx45 phosphorylation, in either Cx45 transfectants or in 293 or BHK21 cells. The addition of the tumor-promoting agent phorbol 12-myristate 13-acetate (TPA) led to an increased 32P phosphate incorporation into the Cx45 protein in transfected cells.
The Cx45 protein was found in homogenates of embryonic brain, kidney, and skin, as well as of adult lung. In kidney of four-day-old mice, Cx45 was detected in glomeruli and distal tubules, whereas connexin32 and −26 were coexpressed in proximal tubules. No connexin43 protein was detected in renal tubules and glomeruli at this stage of development. Our results suggest that cells in proximal and distal tubules are interconnected by gap junction channels made of different connexin proteins. The Cx45 antibodies characterized in this paper should be useful for investigations of Cx45 in renal gap junctional communication.
The effects of a novel membrane-penetrable modulator, 2APB (2-aminoethoxy diphenyl borate), on Ins(1,4,5)P3-induced Ca2+ release were examined. 2APB inhibited Ins(1,4,5)P3-induced Ca2+ release from rat cerebellar microsomal preparations without affecting [3H]Ins(1,4,5)P3 binding to its receptor. The IC50 value (concentration producing 50% inhibition) of 2APB for inhibition of Ins(1,4,5)P3 (100 nM) induced Ca2+ release was 42 microM. Further increase in the concentration of 2APB (more than 90 microM) caused a gradual release of Ca2+ from cerebellar microsomal preparations. Addition of 2APB to the extracellular environment inhibited the cytosolic Ca2+ ([Ca2+]c) rise in intact cells such as human platelets and neutrophils stimulated by thromboxane-mimetic STA2 or thrombin, and leukotriene B4 (LTB4) or formyl-methionine-leucine-phenylalanine (FMLP), respectively. 2APB inhibited the contraction of thoracic aorta isolated from rabbits induced by angiotensin II (AII), STA2, and norepinephrine in a non-competitive manner, but showed no effect on the contraction of potassium-depolarized muscle. 2APB had no effect on the Ca2+ release from the ryanodine-sensitive Ca2+ store prepared from rat leg skeletal muscle and heart. Although the specificity of 2APB with respect to the intracellular signaling system was not fully established, 2APB is the first candidate for a membrane-penetrable modulator of Ins(1,4,5)P3 receptor, and it should be a useful tool to investigate the physiological role of the Ins(1,4,5)P3 receptor in various cells.
Gap junctions are conductive channels that connect the interiors of coupled cells. We determined whether gap junctions propagate transcellular signals during metabolic stress and whether such signaling exacerbates cell injury. Although overexpression of the human proto-oncogene bcl2 in C6 glioma cells normally increased their resistance to injury, the relative resistance of bcl2+ cells to calcium overload, oxidative stress and metabolic inhibition was compromised when they formed gap junctions with more vulnerable cells. The likelihood of death was in direct proportion to the number and density of gap junctions with their less resistant neighbors. Thus, dying glia killed neighboring cells that would otherwise have escaped injury. This process of glial 'fratricide' may provide a basis for the secondary propagation of brain injury in cerebral ischemia.
1. The macroscopic and single channel gating characteristics of connexin (Cx) 50 gap junction channels between pairs of N2A neuroblastoma cells transfected with mouse Cx50 DNA were investigated using the dual whole-cell voltage clamp technique. 2. The macroscopic junctional current (Ij) of Cx50-transfected cells decayed exponentially with time in response to transjunctional voltage (Vj) steps (time constant (tau) of approximately 4 s at a Vj of 30-40 mV and 100-200 ms at a Vj of 80-100 mV). The steady-state junctional conductance (gj) was well described by a two-state Boltzmann equation. The half-inactivation voltage (V0), the ratio of minimal to maximal gj (gmin/gmax) and the equivalent gating charge were +/- 37 mV, 0.21 and 4, respectively. 3. The conductance of single Cx50 channels measured using patch pipettes containing 130 mM CsCl was 220 +/- 13.1 pS (12 cell pairs). A prominent residual or subconductance state corresponding to 43 +/- 4. 2 pS (10 cell pairs) was also observed at large Vj s. 4. The relationship between channel open probability (Po) and Vj was well described by a Boltzmann relationship with parameters similar to those obtained for macroscopic gj (V0 = 34 mV, gating charge = 4.25, maximum P= 0.98). The ensemble average of single channel currents at Vj = 50 mV declined in a monoexponential manner (tau = 905 ms), a value similar to the decline of the macroscopic Ij of Cx50 channels at the same voltage. 5. Ion substitution experiments indicated that Cx50 channels have a lower permeability to anions than to cations (transjunctional conductance of KCl vs. potassium glutamate (gammaj, KCl/gammaj,KGlut), 1.2; 6 cell pairs). 6. The results have important implications for understanding the role of connexins in tissues where Cx50 is a major gap junction component, including the lens.
These studies were performed to evaluate the effect of 2-aminoethoxydiphenyl borate (2-APB), a novel membrane-permeable inositol 1,4,5-trisphosphate-receptor inhibitor on agonist-induced, spontaneous, and KCl-stimulated in vitro myometrial contractions. 2-APB significantly inhibited spontaneous myometrial contractions as well as phasic contractions induced by various uterotonic agonists. Confiriming its effects on intracellular calcium release, 2-APB inhibited phasic contractions in the absence of extracellular calcium. 2-APB had little effect on the tonic response to KCl stimulation, implicating its insignificant effect on voltage-gated calcium channels. The inhibitory effect of 2-APB on phasic contractions was completely reversed by washout. In summary, 2-APB effectively penetrated uterine tissue and significantly inhibited myometrial events previously shown to be mediated through activation of the PI-signaling pathway.
The effect of fenamates on gap junctional intercellular communication was investigated in monolayers of normal rat kidney (NRK) fibroblasts and of SKHep1 cells overexpressing the gap junction protein connexin43 (Cx43). Using two different methods to study gap junctional intercellular communication, single electrode voltage-clamp step response measurements and dye microinjection, we show that fenamates are reversible blockers of Cx43-mediated intercellular communication. After adding fenamates to a confluent monolayer of electrically coupled NRK fibroblasts, the voltage step-induced capacitive current transient changed from a transient characteristic for charging multiple coupled cell capacitances to one characteristic for a single cell in isolation. The capacitance of completely uncoupled cells was 19.7 +/- 1.0 pF (mean +/- S.E.M.; n = 11). Junctional conductance between the patched cell and the surrounding cells in the monolayer changed from >140.7 +/- 9.6 nS (mean +/- S.E.M.; n = 14) to <1.4 +/- 0.4 nS (mean +/- S.E.M.; n = 11) after uncoupling. Electrical coupling could be restored to >51.8 +/- 4.2 nS (mean +/- S.E.M.; n = 11) by washout of the fenamates. Voltage-clamp step response measurements showed that the potency of fenamates in inhibiting electrical coupling decreases in the order meclofenamic acid > niflumic acid > flufenamic acid. The half-maximal concentration determined by dye-coupling experiments was 25 and 40 microM for meclofenamic acid and flufenamic acid, respectively. Inhibition of gap junctional communication by fenamates did not involve changes in intracellular calcium or pH, and was unrelated to protein kinase C activity or an inhibition of cyclooxygenase activity. Voltage-clamp step response measurements in confluent monolayers of SKHep1 cells that had been stably transfected with Cx43 revealed that fenamates are potent blockers of Cx43-mediated intercellular communication. In conclusion, fenamates represent a novel class of reversible gap junction blockers that can be used to study the role of Cx43-mediated gap junctional intercellular communication in biological processes.
We demonstrate that the antimalarial drug quinine specifically reduces currents through gap junctions formed by some connexins (Cx) in transfected mammalian cells, but does not affect other gap junction types. Quinine blocked Cx36 and Cx50 junctional currents in a reversible and concentration-dependent manner with half maximal blocking concentrations of 32 and 73 microM, respectively; Hill coefficients for block by quinine were about 2 for both connexins. In contrast, quinine did not substantially block gap junction channels formed by Cx26, Cx32, Cx40, and Cx43, and only moderately affected Cx45 junctions. To determine the location of the binding site of quinine (pKa = 8.7), we investigated the effect of quinine at various external and internal pH values and the effect of a permanently charged quaternary derivative of quinine. Our results indicate that the binding site for quinine is intracellular, possibly within the pore. Single-channel studies indicated that exposure to quinine induced slow transitions between open and fully closed states that decreased open probability of the channel. Quinine thus offers a potentially useful method to block certain types of gap junction channels, including those between neurons that are formed by Cx36. Moreover, quinine derivatives that are excluded from other types of membrane channels may provide molecules with connexin-specific as well as connexin-selective blocking activity.
Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca(2+)](i) and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca(2+)](i), with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca(2+)](i) was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca(2+)](i) were abolished by loading the preparations with the Ca(2+) chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP(3) induced Ca(2+) release, by N-ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl(-))-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP(3) formation. This causes Ca(2+) release from intracellular stores which activates Ca(2+)-dependent Cl(-) channels.
Physiological functions of urinary bladder profoundly reflect smooth muscle mechanical activity. Urinary bladder smooth muscle itself produces myogenic rhythmic contraction, and this spontaneous mechanical event could be the fundamental determinant of urinary bladder functions. The spontaneous contraction of urinary bladder smooth muscle is thought to be triggered primarily by the action potential generated in this smooth muscle cell. Modulators of ion channels contributing to the configuration of action potential also affect urinary bladder smooth muscle mechanical activity as expected exactly from the effects on the electrical event. In the present study, we show that the frequency of action potential recorded in intact strip of guinea-pig urinary bladder smooth muscle is dramatically increased by 2-aminoethoxydiphenyl borate (2-APB; 30 microM) from 0.2 Hz to 1 Hz (approximately 500% increments). In contrast to an increasing effect expected from the membrane electrical alterations, mechanical activity (both contraction amplitude and frequency) of this smooth muscle is unexpectedly reduced by the same concentration of 2-APB to approximately 35% of the control. The present results firstly show an apparent dissociation of electrical-mechanical coupling in urinary bladder smooth muscle. The alteration of membrane electrical activity might not be the exclusive trigger mechanism responsible for the generation of spontaneous rhythmic contraction of this smooth muscle.
Connexin-null mice and human genetic gap junction diseases illustrate the important roles that gap junction channels play under normal conditions, and the neuro- and cardioprotective effects of gap junction blocking agents demonstrate that closure of these channels may be beneficial in certain pathological situations. This overview summarizes studies in which gap junction modifying reagents have been characterized, highlighting examples of agents for which selectivity for gap junction subtypes has been demonstrated. In addition, strategies for targeting connexin domains through peptide inhibitors are outlined, which may ultimately provide agents that are not only connexin-selective in their actions, but also affect only a subset of a gap junction channel's gating responses.
The effects of spermine and spermidine, endogenous polyamines that block many forms of ion channels, were investigated in homotypic connexin (Cx)-40 gap junctions expressed in N2A cells. Spermine blocked up to 95% of I(j) through homotypic Cx40 gap junctions in a concentration- and transjunctional voltage (V(j))-dependent manner. V(j) was varied from 5 to 50 mV in 5-mV steps and the dissociation constants (K(m)) were determined from spermine concentrations ranging from 10 micro M to 2 mM. The K(m) values ranged from 4.9 mM to 107 micro M for 8.6 < or = V(j) < or = 37.7 mV, within the physiological range of intracellular spermine for V(j) > or = 20 mV. The K(m) values for spermidine were > or = 5 mM. Estimates of the electrical distance (delta) for spermine (z = +4) and spermidine (z = +3) were 0.96 and 0.76 respectively. Cx40 single channel conductance was 129 pS in the presence of 2-mM spermine and channel open probability was significantly reduced in a V(j)-dependent manner. Similar concentrations of spermine did not block I(j) through homotypic Cx43 gap junctions, indicating that spermine selectively blocks Cx40 gap junctions. This is contrary to our previous findings that large tetraalkylammonium ions, also known to block several forms of ion channels, block junctional currents (I(j)) through homotypic connexin Cx40 and Cx43 gap junctions.
We determined the effect of flufenamic acid (FFA) and related derivatives on gap junction channel currents, applying the dual whole-cell patch-clamp technique to pairs of N2A neuroblastoma cells transfected with various connexins. FFA reduced gap junction channel currents in a reversible and concentration-dependent manner. Half-maximal concentrations for FFA-induced reduction of junctional conductance in cell pairs coupled by different connexins were similar (20 to 60 microM), indicating that FFA does not greatly discriminate between connexin subtypes. Hill coefficients for blockade were approximately 3, indicating a high degree of cooperativity. Analogs of FFA also reduced junctional conductance with similar potencies, whereas other unrelated chloride channel blockers had no effect. Inhibition of gap junction channels by FFA (pKa approximately 3.8) was increased at low external pH, suggesting that the uncharged form of the drug is important for blockade. The effect of FFA did not seem to be mediated by direct binding of the drug to the pore of the gap junction channel. Internal application of high concentrations of FFA by addition to patch pipettes did not cause inhibition of channel currents. The magnitude of inhibition was neither voltage-dependent nor influenced by the nature of permeant ion. Single-channel recordings indicated that FFA reduced the channel-open probability without modifying the current amplitude and induced slow transitions between open and closed states. We propose that FFA inhibits gap junctions by inducing a conformational change in the protein upon binding to a site that is presumably located within the membrane.
The membrane-permeable, noncompetitive inositol 1,4,5-trisphosphate (IP3)-receptor inhibitor 2-aminoethoxydiphenyl borane (2-APB) has been widely used to probe for IP3-receptor involvement in calcium signaling pathways. However, a number of recent studies in different cell types revealed other sites of action of 2-APB. In this study, we examined the influence of 2-APB on capacitative calcium entry and intracellular Ca2+ concentrations in rat basophilic leukemia (RBL-2H3 m1) cells. 2-APB was found to inhibit capacitative calcium entry, but at concentrations greater than 50 microM, a new effect of 2-APB was observed. When capacitative calcium entry was blocked with Gd3+, 2-APB caused an increase in cytoplasmic Ca2+. This increase in intracellular Ca2+ was not caused by altered buffering of cytoplasmic Ca2+ and was not caused by or in any way affected by the depletion of intracellular Ca2+ stores. Associated with the increase in intracellular Ca2+, in the presence of 2 mM Ca2+, 2-APB activated single channels in the plasma membrane with a conductance of approximately 50 pS. These channels seem to be nonselective cation channels; monovalent cations are the major carriers of current, but finite permeability to Ca2+ leads to a significant intracellular Ca2+ signal. Experiments with excised patches indicate that 2-APB activates these channels from the outer aspect of the cell membrane. This effect of 2-APB further illustrates the complex actions of this compound and reveals the presence in RBL-2H3 m1 cells of a novel, ligand-gated calcium-permeable channel.
It has previously been suggested that volume-regulated anion channels (VRACs) and store-operated channels (SOCs) interact with each other according to their expected colocalization in the plasma membrane of LNCaP cells. In order to study interactions between these two channels, we used 2-aminoethoxydiphenyl borate (2-APB) as a regular SOC inhibitor. Surprisingly 2-APB reduced VRAC activity in a dose-dependent manner (IC(50)=122.8 microM), but not 2,2-diphenyltetrahydrofuran (a structural analog of 2-APB). This effect was also present in keratinocytes. We conclude that 2-APB is an inhibitor of the VRAC family, and is also a potent tool to study the SOC-VRAC interaction in LNCaP cells.
Gap junctions are intercellular channels which directly connect the cytoplasm between neighboring cells. In the central nervous system (CNS) various kinds of cells are coupled by gap junctions, which play an important role in maintaining normal function. Neuronal gap junctions are involved in electrical coupling and may also contribute to the recovery of function after cell injury. Astrocytes are involved in the pathology of most neuronal disorders, including brain ischemia, Alzheimer's disease and epilepsy. In the pathology of brain tumors, gap junctions may be related to the degree of malignancy and metastasis. However, the role of connexins, gap junctions and hemichannels in the pathology of the diseases in the CNS is still ambiguous. Of increasing importance is the unraveling of the function of gap junctions in the neural cell network, involving neurons, astrocytes, microglia and oligodendrocytes. A better understanding of the role of gap junctions may contribute to the development of new therapeutic approaches to treating diseases of the CNS.
The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels
that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members,
TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception
to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3
are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2,
and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and
TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons,
and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid.
Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic
target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation.
To investigate mechanisms underlying the transmission of spontaneous Ca2+ signals in the bladder, changes in intracellular concentrations of Ca2+ ([Ca2+]i) were visualized in isolated detrusor smooth muscle bundles of the guinea-pig urinary bladder loaded with a fluorescent Ca2+ indicator, fura-PE3 or fluo-4. Spontaneous increases in [Ca2+]i (Ca2+ transients) preferentially originated along the boundary of muscle bundles and then spread to the other boundary (Ca2+ waves). The synchronicity of Ca2+ waves across the bundles was disrupted by 18beta-glycyrrhetinic acid (18beta-GA, 40 microm), carbenoxolone (30 microm) or 2-aminoethoxydiphenylborate (2-APB, 50-100 microm), while CPA (10 microm), ryanodine (100 microm), xestospongin C (3 microm) and U-73122 (10 microm) had no effect. Intracellular recordings using two independent microelectrodes demonstrated that 2-APB (100 microm) blocked electrical coupling between detrusor smooth muscle cells. Nifedipine (10 microm) but not nominal Ca2+-free solution diminished the synchronicity of Ca2+ waves before preventing their generation. Staining for c-kit identified interstitial cells (IC) located along both boundaries of muscle bundles. IC were also scattered amongst smooth muscle cells and were more dominantly distributed in connective tissue between muscle bundles. IC generated nifedipine-resistant spontaneous Ca2+ transients, which occurred independently of those of smooth muscles. In conclusion, the propagation of Ca2+ transients in the bladder appears to be exclusively mediated by the spread of action potentials through gap junctions being facilitated by the regenerative nature of L-type Ca2+ channels, without significant contribution of intracellular Ca2+ stores. IC in the bladder may modulate the transmission of Ca2+ transients originating from smooth muscle cells rather than being the pacemaker of spontaneous activity.
In this article we overview the most important antiarrhythmic and positive inotropic mechanisms based on pharmacological modification of an ion channel or a transport protein in the surface membrane of cardiac myocytes. First we briefly characterize the ion currents mediated by these proteins in atrial and ventricular cells. Since the level of expression of ion channels is markedly altered in various types of chronic heart diseases, such as atrial fibrillation or heart failure, cardiac remodelling characteristic of these cases is also discussed. The paper gives evaluation of the currently applied most important antiarrhythmic strategies and some insight into the perspectives of the future by reviewing a few but promising mechanisms and drugs that are currently investigated. Positive inotropic agents and mechanisms are similarly treated, focusing primarily on proarrhythmic risks or potential antiarrhythmic effects of these compounds. Based on the backgrounds and aims above, modification of the followings factors is discussed in details: I(Na), I(Ca), I(Kr), I(Ks), I(Kl), I(to), I(Kur), I(K,Ach), I(K,ATP), I(f), gap-junction channel, Na(+)/K(+) pump, Na(+)/Ca(2+) exchanger, Na(+)/H(+) exchanger, as well as the intracellular concentrations of sodium and calcium ions. In addition to the critical evaluation of each manipulation, the following general conclusions can be drawn. (1) Since large modifications in action potential parameters are usually disadvantageous at long time scale, combination of the various mechanisms, each represented at a moderate degree, appears to be better. (2) Regarding Class III. antiarrhythmic action, selective potassium channel blockers free of reverse rate-dependent properties should be preferred. (3) Partial inhibition of the Na(+)/Ca(2+) exchanger may result-paradoxically in an antiarrhythmic action under specific conditions, in addition to its positive inotropic effect. We believe that investigation of new antiarrhythmic mechanisms, rather than new compounds of the old families, might be most beneficial in order to effectively treat life threatening cardiac arrhythmias in the future.
2-aminoethoxydiphenyl borate (2-APB) has been widely used as a blocker of the IP3 receptor and TRP channels, including store-operated calcium channels. We now show in monolayers of normal rat kidney cells (NRK/49F) that 2-APB completely and reversibly blocks gap junctional intercellular communication at concentrations similar to that required for inhibition of PGF2alpha-induced increases in intracellular calcium. Gap junctional conductances between NRK cells were estimated with single-electrode patch-clamp measurements and were fully blocked by 2-APB (50 microM), when applied extracellularly but not via the patch pipette. Half maximal inhibition (IC50) of electrical coupling in NRK cells was achieved at 5.7 microM. Similar results were obtained for human embryonic kidney epithelial cells (HEK293/tsA201) with an IC50 of 10.3 microM. Using 2-APB as an electrical uncoupler of monolayer cells, we could thus measure inward rectifier potassium, L-type calcium, and calcium-dependent chloride membrane currents in confluent NRK monolayers, with properties similar to those in dissociated NRK cells in the absence of 2-APB. The electrical uncoupling action described here is a new 2-APB property that promises to provide a powerful pharmacological tool to study single-cell properties in cultured confluent monolayers and intact tissues by electrical and chemical uncoupling of the cells without the need of prior dissociation.
2‐Aminoethoxydiphenyl borate (2‐APB) has been widely used to examine the roles of inositol 1,4,5‐trisphosphate receptors (IP 3 Rs) and store‐operated Ca ²⁺ entry and is an emerging modulator of cationic channels encoded by transient receptor potential (TRP) genes.
Using Ca ²⁺ ‐indicator dye and patch‐clamp recording we first examined the blocking effect of 2‐APB on human TRPC5 channels expressed in HEK‐293 cells.
The concentration–response curve has an IC 50 of 20 μ M and slope close to 1.0, suggesting one 2‐APB molecule binds per channel. The blocking effect is not shared by other Ca ²⁺ channel blockers including methoxyverapamil, nifedipine, N ‐propargylnitrendipine, or berberine.
In whole‐cell and excised membrane patch recordings, 2‐APB acts from the extracellular but not intracellular face of the membrane.
Block of TRPC5 by 2‐APB is less at positive voltages, suggesting that it enters the electric field or acts by modulating channel gating.
2‐APB also blocks TRPC6 and TRPM3 expressed in HEK‐293 cells, but not TRPM2.
Block of TRP channels by 2‐APB may be relevant to cell proliferation because 2‐APB has a greater inhibitory effect on proliferation in cells overexpressing TRPC5.
Our data indicate a specific and functionally important binding site on TRPC5 that enables block by 2‐APB. The site is only available via an extracellular route and the block shows mild voltage‐dependence.
British Journal of Pharmacology (2005) 145 , 405–414. doi: 10.1038/sj.bjp.0706197
The action of 2-aminoethoxydiphenyl borate (2-APB) on Ca(2+) signalling in HeLa cells and cardiac myocytes was investigated. Consistent with other studies, we found that superfusion of cells with 2-APB rapidly inhibited inositol 1,4,5-trisphosphate (InsP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOC). In addition to abrogating hormone-evoked Ca(2+) responses, 2-APB could antagonise Ca(2+) signals evoked by a membrane permeant InsP(3) ester. 2-APB also slowed the recovery of intracellular Ca(2+) signals consistent with an effect on Ca(2+) ATPases. The inhibitory action of 2-APB on InsP(3) receptors (InsP(3)Rs), SOC channels and Ca(2+) pumps persisted for several minutes after washout of the compound. Application of 2-APB to unstimulated cells had no effect on subsequent Ca(2+) responses suggesting that it has a use-dependent action. Mitochondria in cells treated with 2-APB showed a rapid and slowly reversible swelling. 2-APB did not cause the mitochondria to depolarise, but it reduced the extent of mitochondrial calcium uptake. Although 2-APB has been demonstrated not to affect voltage-operated Ca(2+) channels or ryanodine receptors, we found that it gave a concentration-dependent long-lasting inhibition of Ca(2+) signalling in electrically-stimulated cardiac myocytes, where InsP(3)Rs and SOC channels do not play a significant role. Our data suggest that 2-APB has multiple cellular targets, a use-dependent action, is difficult to reverse and may affect Ca(2+) signalling in cell types where InsP(3) and SOC are not active.