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Depletion of intracellular Ca2+ stores activates a Ca2+-selective channel in vascular endothelium
Abstract
The present study was designed to identify the channel responsible for Ca2+ influx after depletion of intracellular Ca2+ stores. Different maneuvers that deplete intracellular Ca2+ stores activated a Ca(2+)-selective channel. Superfusion of single bovine aortic endothelial cells with 50 nmol/l bradykinin, 10 mumol/l ATP, or 10 mumol/l 2,5-di(tert-butyl)-1,4-benzohydroquinone produced activation of channels of the same amplitude in cell-attached patches. Channel activity declined within the first minute after patch excision. The channel showed strong inward rectification and a reversal potential of 0 mV in symmetrical sodium sulfate (Na2SO4) solution. Under these conditions, the conductance was 5 pS in the inward direction. Addition of 10 mmol/l Ca2+ to the extracellular solution shifted the reversal potential to +30 +/- 5 mV, and the conductance for inward current was 11 pS. The reversal potential was used to calculate an ion permeability ratio of Ca2+/Na+ > 10:1.
... A flurry of investigations published over the last twenty years paved the way towards the general agreement that, in vascular endothelial cells, Stromal Interaction Molecule 1 (STIM1) and Orai1 proteins mediate the I CRAC [38][39][40], whereas members of the Transient Receptor Potential (TRP) Canonical (TRPC) subfamily of non-selective cation channels underlie the I SOC [41][42][43]. A careful scrutiny of the literature, however, reveals that depletion of the ER Ca 2+ store can activate a third endothelial Ca 2+ -entry pathway, which is more Ca 2+ selective than the I SOC but displays a weaker inward rectification and has a more negative reversal potential (E rev ) as respect to the I CRAC [44][45][46][47]. This current has been termed as I SOC [48] or I CRAC -like [46,49]. ...
... The search for the endothelial I CRAC led to the discovery that ER Ca 2+ depletion by stimuli that do not involve InsP 3 -induced Ca 2+ release, i.e., the SERCA inhibitors CPA, thapsigargin, and tBHQ, induced either an I SOC [47,187,188] or an I CRAC -like current [44,189]. In the present section, we discuss the evidence supporting the notion that the endothelial I SOC is mediated by STIM1, TRPC1, and TRPC4, while in Section 6 we illustrate the findings indicating that the incorporation of Orai1 into this supermolecular complex turns the I SOC into an I CRAC -like current. ...
... Some of the earlier reports of the endothelial I SOC described a membrane current that actually displayed intermediate electrophysiological features between I SOC and I CRAC (Table 7) [49]. Vaca and Kunze showed that, in BAECs, tBHQ elicited a non-selective cation current that displayed an inwardly-rectifying IV relationship, presented a single-channel conductance of 5 pS between −40 and −80 mV, reversed at ≈+6 mV, and could also be activated in response to physiological depletion of the InsP 3 -sensitive ER Ca 2+ pool with ATP or bradykinin (Table 7) [44,189]. Subsequently, the Stevens group showed that thapsigargin activated an inwardly-rectifying cation current, which reversed between +30 mV and +40 mV and presented Ca 2+ -dependent inactivation, in rPAECs (Table 7) [69,140,220,221]. ...
Store-operated Ca2+ entry (SOCE) is activated in response to the inositol-1,4,5-trisphosphate (InsP3)-dependent depletion of the endoplasmic reticulum (ER) Ca2+ store and represents a ubiquitous mode of Ca2+ influx. In vascular endothelial cells, SOCE regulates a plethora of functions that maintain cardiovascular homeostasis, such as angiogenesis, vascular tone, vascular permeability, platelet aggregation, and monocyte adhesion. The molecular mechanisms responsible for SOCE activation in vascular endothelial cells have engendered a long-lasting controversy. Traditionally, it has been assumed that the endothelial SOCE is mediated by two distinct ion channel signalplexes, i.e., STIM1/Orai1 and STIM1/Transient Receptor Potential Canonical 1(TRPC1)/TRPC4. However, recent evidence has shown that Orai1 can assemble with TRPC1 and TRPC4 to form a non-selective cation channel with intermediate electrophysiological features. Herein, we aim at bringing order to the distinct mechanisms that mediate endothelial SOCE in the vascular tree from multiple species (e.g., human, mouse, rat, and bovine). We propose that three distinct currents can mediate SOCE in vascular endothelial cells: (1) the Ca2+-selective Ca2+-release activated Ca2+ current (ICRAC), which is mediated by STIM1 and Orai1; (2) the store-operated non-selective current (ISOC), which is mediated by STIM1, TRPC1, and TRPC4; and (3) the moderately Ca2+-selective, ICRAC-like current, which is mediated by STIM1, TRPC1, TRPC4, and Orai1.
... However, with the intracellular Ca 2+ elevates, the relative high concentration of Ca 2+ at micromolar instead inhibits the opening of IP3Rs and RyRs. On the other hand, the emptying Ca 2+ store activates the extracellular Ca 2+ influx through plasma membrane Ca 2+ channels to replenish Ca 2+ in the endoplasmic reticulum, a process known as store-operated Ca 2+ entry (SOCE) (Oike et al., 1994;Vaca and Kunze, 1994). In endothelial cells, it has been reported that Ca 2+ -store release and SOCE are required for the regulation of endothelial permeability or proliferation (Abdullaev et al., 2008;Oike et al., 1994;Sun et al., 2017;Vaca and Kunze, 1994), further some Ca 2+ store-related channels or transmembrane proteins located in the endoplasmic reticulum are identified for the regulation of EC migration during vascular morphogenesis (e.g., two-pore channels, TMEM33) (Favia et al., 2014;Savage et al., 2019). ...
... On the other hand, the emptying Ca 2+ store activates the extracellular Ca 2+ influx through plasma membrane Ca 2+ channels to replenish Ca 2+ in the endoplasmic reticulum, a process known as store-operated Ca 2+ entry (SOCE) (Oike et al., 1994;Vaca and Kunze, 1994). In endothelial cells, it has been reported that Ca 2+ -store release and SOCE are required for the regulation of endothelial permeability or proliferation (Abdullaev et al., 2008;Oike et al., 1994;Sun et al., 2017;Vaca and Kunze, 1994), further some Ca 2+ store-related channels or transmembrane proteins located in the endoplasmic reticulum are identified for the regulation of EC migration during vascular morphogenesis (e.g., two-pore channels, TMEM33) (Favia et al., 2014;Savage et al., 2019). In parallel, previous study has shown that Ca 2+ influx in ECs is required for EC proliferation or vessel invasion (Kohn et al., 1995), and our previous evidence also shows that Ca 2+ activities in ETCs mediated by the mechanosensitive cationic channel Piezo1 plays a crucial role in vascular pathfinding (Liu et al., 2020). ...
Endothelial tip cells (ETCs) located at growing blood vessels display high morphological dynamics and associated intracellular Ca²⁺ activities with different spatiotemporal patterns during migration. Examining the Ca²⁺ activity and morphological dynamics of ETCs will provide an insight for understanding the mechanism of vascular development in organs, including the brain. Here, we describe a method for simultaneous monitoring and relevant analysis of the Ca²⁺ activity and morphology of growing brain ETCs in larval zebrafish.
For complete details on the use and execution of this protocol, please refer to Liu et al. (2020).
... TRPC channels show a distinct repertoire of biophysical properties as compared to Orai1, which result in a store-operated current, also termed ISOC, which is featured by: 1000 times higher unitary conductance (in the pS range), linear I-V relationship and Erev of ≈ 0 mV as TRPC channels do not discriminate between Na + , K + and Ca 2+ [145,146]. Quite surprisingly, however, electrophysiological recordings revealed that the endothelial TRPC1 and TRPC4 channels mediate either an ICRAC-like current [141] or a less Ca 2+ -selective current with biophysical properties intermediate between ICRAC and ISOC [142,143,147,148]. Alternately, endothelial TRPC channels may line the pore of an ISOC-like conductance [124,[149][150][151]. ...
It has long been known that endothelial Ca2+ signals drive angiogenesis by recruiting multiple Ca2+-sensitive decoders in response to pro-angiogenic cues, such as vascular endothelial growth factor, basic fibroblast growth factor, stromal derived factor-1α and angiopoietins. Recently, it was shown that intracellular Ca2+ signaling also drives vasculogenesis by stimulation proliferation, tube formation and neovessel formation in endothelial progenitor cells. Herein, we survey how growth factors, chemokines and angiogenic modulators use endothelial Ca2+ signaling to regulate angiogenesis and vasculogenesis. The endothelial Ca2+ response to pro-angiogenic cues may adopt different waveforms, ranging from Ca2+ transients or biphasic Ca2+ signals to repetitive Ca2+ oscillations, and is mainly driven by endogenous Ca2+ release through inositol-1,4,5-trisphosphate receptors and by store-operated Ca2+ entry through Orai1 channels. Lysosomal Ca2+ release through nicotinic acid adenine dinucleotide phosphate-gated two-pore channels is, however, emerging as a crucial pro-angiogenic pathway, which sustains intracellular Ca2+ mobilization. Understanding how endothelial Ca2+ signaling regulates angiogenesis and vasculogenesis could shed light on alternative strategies to induce therapeutic angiogenesis or interfere with the aberrant vascularization featuring cancer and intraocular disorders.
... ECs are generally thought to be non-excitable and as such utilise store-operated calcium entry (SOCE) as their primary means to maintain Ca 2+ levels in the ER after influx 13,14 . The principal trigger for SOCE activation occurs when intracellular calcium stores with the ER are depleted and these are refilled via interaction of TRPC1 and ORAI1 on the plasma membrane with ER-resident STIM1, which form the calcium-release activated channel [15][16][17][18] . ...
Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks. Vascular endothelial growth factor (VEGF) is essential for angiogenesis and induces downstream signalling pathways including increased cytosolic calcium levels. Here we show that transmembrane protein 33 (tmem33), which has no known function in multicellular organisms, is essential to mediate effects of VEGF in both zebrafish and human ECs. We find that tmem33 localises to the endoplasmic reticulum in zebrafish ECs and is required for cytosolic calcium oscillations in response to Vegfa. tmem33-mediated endothelial calcium oscillations are critical for formation of endothelial tip cell filopodia and EC migration. Global or endothelial-cell-specific knockdown of tmem33 impairs multiple downstream effects of VEGF including ERK phosphorylation, Notch signalling and embryonic vascular development. These studies reveal a hitherto unsuspected role for tmem33 and calcium oscillations in the regulation of vascular development.
... ECs are generally thought to be non-excitable and as such utilise store-operated calcium entry (SOCE) as their primary means to maintain Ca 2+ levels in the ER after influx 13,14 . The principal trigger for SOCE activation occurs when intracellular calcium stores with the ER are depleted and these are refilled via interaction of TRPC1 and ORAI1 on the plasma membrane with ER-resident STIM1, which form the calcium-release activated channel [15][16][17][18] . ...
Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks. Vascular endothelial growth factor (VEGF) is essential for angiogenesis and induces downstream signalling pathways including increased cytosolic calcium levels. Here we show that transmembrane protein 33 (tmem33), which has no known function in multicellular organisms, is essential to mediate effects of VEGF in both zebrafish and human ECs. We find that tmem33 localises to the endoplasmic reticulum in zebrafish ECs and is required for cytosolic calcium oscillations in response to Vegfa. tmem33-mediated endothelial calcium oscillations are critical for formation of endothelial tip cell filopodia and EC migration. Global or endothelial-cell-specific knockdown of tmem33 impairs multiple downstream effects of VEGF including ERK phosphorylation, Notch signalling and embryonic vascular development. These studies reveal a hitherto unsuspected role for tmem33 and calcium oscillations in the regulation of vascular development.
Patch-clamp experiments combined with indo-1 measurement of free intracellular Ca²⁺ concentration ([Ca²⁺]i) were used to determine the homeostatic systems involved in the maintenance of resting [Ca²⁺]i and the clearance of Ca²⁺ transients following activation of voltage-gated Ca²⁺ channels in neurones from rat superior cervical ganglion (SCG). In these neurones, the Ca²⁺ buffering capacity was estimated to be ≈ 1000 at [Ca²⁺]i close to rest and ≈ 250 at [Ca²⁺]i ≈ 1 μM and to involve at least two buffering systems with different affinities for Ca²⁺. Removal of extracellular Ca²⁺ led to a decrease in [Ca²⁺]i that was mimicked by the addition of La³⁺. This decrease in [Ca²⁺]i was more pronounced after inhibition of the endoplasmic reticulum Ca²⁺ uptake system (SERCA) and subsequent depletion of the intracellular stores. Inhibition of the plasma membrane Ca2+ pump (PMCA) by intracellular carboxyeosin or extracellular alkalisation (pH 9) both increased resting [Ca²⁺]i and prolonged the recovery of Ca²⁺ transients at peak [Ca²⁺]i < 500 nM. For [Ca²⁺]i loads > 500 nM, recovery showed an additional plateau phase that was abolished in carbonyl cyanide m-chlorohydrazone (CCCP) or on omitting Na⁺ from the intracellular solution. Inhibition of the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) and of SERCA had a small but significant effect on the rate of decay of these larger Ca²⁺ transients. However, neither of these mechanisms appeared to contribute substantially to the recovery from rises in [Ca²⁺]i below 1 μM. In conclusion, resting [Ca²⁺]i is maintained as the result of a passive Ca²⁺ influx regulated by a large Ca²⁺ buffering system, Ca²⁺ extrusion via a PMCA and Ca²⁺ transport from the intracellular stores. PMCA is also the principal Ca²⁺ extrusion system at low Ca²⁺ loads, with additional participation of the NCX and intracellular organelles at high [Ca²⁺]i.
Endothelial cells (EC) form a unique signal-transducing surface in the vascular system. The abundance of ion channels in the plasma membrane of these nonexcitable cells has raised questions about their functional role. This review presents evidence for the involvement of ion channels in endothelial cell functions controlled by intracellular Ca ²⁺ signals, such as the production and release of many vasoactive factors, e.g., nitric oxide and PGI 2 . In addition, ion channels may be involved in the regulation of the traffic of macromolecules by endocytosis, transcytosis, the biosynthetic-secretory pathway, and exocytosis, e.g., tissue factor pathway inhibitor, von Willebrand factor, and tissue plasminogen activator. Ion channels are also involved in controlling intercellular permeability, EC proliferation, and angiogenesis. These functions are supported or triggered via ion channels, which either provide Ca ²⁺ -entry pathways or stabilize the driving force for Ca ²⁺ influx through these pathways. These Ca ²⁺ -entry pathways comprise agonist-activated nonselective Ca ²⁺ -permeable cation channels, cyclic nucleotide-activated nonselective cation channels, and store-operated Ca ²⁺ channels or capacitative Ca ²⁺ entry. At least some of these channels appear to be expressed by genes of the trp family. The driving force for Ca ²⁺ entry is mainly controlled by large-conductance Ca ²⁺ -dependent BK Ca channels ( slo), inwardly rectifying K ⁺ channels (Kir2.1), and at least two types of Cl ⁻ channels, i.e., the Ca ²⁺ -activated Cl ⁻ channel and the housekeeping, volume-regulated anion channel (VRAC). In addition to their essential function in Ca ²⁺ signaling, VRAC channels are multifunctional, operate as a transport pathway for amino acids and organic osmolytes, and are possibly involved in endothelial cell proliferation and angiogenesis. Finally, we have also highlighted the role of ion channels as mechanosensors in EC. Plasmalemmal ion channels may signal rapid changes in hemodynamic forces, such as shear stress and biaxial tensile stress, but also changes in cell shape and cell volume to the cytoskeleton and the intracellular machinery for metabolite traffic and gene expression.
Calcium entry pathways in non-excitable cells presents a concise synthesis of thoughtfully selected topics covering from the different calcium entry mechanisms in non-excitable cells to the cellular microdomains and organelles regulating the calcium entry process. Particular attention is given to the fascinating group of ion channels involved in different calcium entry pathways as well as the emerging role of these channels in human disease. Calcium entry is an essential mechanism for cellular function in non-excitable cells. In general, two main calcium entry pathways exist in non-excitable cells: one pathway, named store-operated calcium entry (SOCE) requires store depletion and the second pathway is regulated by receptor occupation, but independently on calcium store depletion. The search for the molecular components of calcium entry has identified the stromal interaction molecule 1 (STIM1), as the calcium sensor of the intracellular calcium stores, and Orai as well as TRP channels as the calcium-permeable channels located in the plasma membrane. The location, interactions and function of these channels are finely regulated by a number of scaffolding proteins, membrane microdomains and cellular organelles that fine tune the amount of calcium entering the cell. Cutting-edge and user-friendly, this volume presents relevant background information, critical analysis of the current observations and directions for future research. The book is intended for basic scientists specializing in cellular biology or ion transport, as well as for biomedical researchers.
The depletion of an inositol 1,4,5-trisphosphate-sensitive intracellular Ca²⁺ pool has been proposed to be the signal for Ca²⁺ entry in agonist-activated cells. Consistent with this idea, thapsigargin, which releases intracellular Ca²⁺ without inositol phosphate formation, has been reported to activate Ca²⁺ entry in certain cells. We now report the effects of thapsigargin on Ca²⁺ entry in parotid acinar cells. In fura-2-loaded parotid acinar cells, thapsigargin caused a sustained elevation of [Ca²⁺]i but did not increase inositol phosphate formation. In the absence of extracellular Ca²⁺, the increase in [Ca²⁺]i was transient, suggesting that thapsigargin activates both the release of Ca²⁺ from intracellular stores and the entry of Ca²⁺ from the extracellular space. In the absence of extracellular Ca²⁺, pretreatment with methacholine, an agonist believed to mobilize Ca²⁺ through the production of inositol 1,4,5-trisphosphate, inhibited but did not completely block the response to thapsigargin; likewise, pretreatment with thapsigargin inhibited the response to methacholine. In permeabilized cells, thapsigargin gradually released Ca²⁺, whereas inositol 1,4,5-trisphosphate caused a rapid and transient discharge of Ca²⁺. The simultaneous addition of thapsigargin with inositol 1,4,5-trisphosphate evoked a maximum Ca²⁺ release similar to that for inositol 1,4,5-trisphosphate alone, but the reuptake seen with inositol 1,4,5-trisphosphate alone was abolished. In intact cells, methacholine and thapsigargin together produced a greater initial release of Ca²⁺ than either alone, but they were not additive in the sustained phase of Ca²⁺ mobilization. These results demonstrate that the mechanisms for activation of Ca²⁺ entry by thapsigargin and methacholine are the same and are consistent with the idea that entry is initiated by the depletion of the intracellular inositol 1,4,5-trisphosphate-sensitive Ca²⁺ pool. The results also indicate that, in contrast to previously proposed models, Ca²⁺ entry into agonist-activated cells occurs directly across the plasma membrane to the cytoplasm rather than through a cycle of uptake and release by the intracellular Ca²⁺ pool.
In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.
Previous studies in non-excitable cells have suggested that depletion of internal Ca2+ stores activates Ca2+ influx from the extracellular space via a mechanism that does not require stimulation of phosphoinositide hydrolysis. To test this hypothesis in vascular endothelial cells, the effect of the Ca(2+)-ATPase/pump inhibitor 2,5-di-t-butylhydroquinone (BHQ) on cytosolic free Ca2+ concentration ([Ca2+]i) was examined. BHQ produced a dose-dependent increase in [Ca2+]i, which remained elevated over basal values for several minutes and was substantially inhibited in the absence of extracellular Ca2+. Application of bradykinin after BHQ demonstrated that the BHQ-sensitive compartment partially overlapped the bradykinin-sensitive store. Similar results were obtained with thapsigargin and cyclopiazonic acid, two other Ca(2+)-ATPase inhibitors. Although BHQ had no effect on phosphoinositide hydrolysis, both 45Ca2+ influx and efflux were stimulated by this agent. These results suggest that depletion of the agonist-sensitive Ca2+ store is sufficient for activation of Ca2+ influx. Several characteristics of the Ca(2+)-influx pathway activated by internal store depletion were compared with those of the agonist-activated pathway. Bradykinin-stimulated Ca2+ influx was increased at alkaline extracellular pH (pHo), and was inhibited by extracellular La3+, by depolarization of the membrane, and by the novel Ca(2+)-influx blocker 1-(beta-[3-(4-methoxyphenyl)propoxy]-4- methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365). Additionally, bradykinin stimulated influx of both 45Ca2+ and 133Ba2+, consistent with the hypothesis that the agonist-activated influx pathway is permeable to both of these bivalent cations. Likewise, activation of Ca2+ influx by BHQ, thapsigargin and cyclopiazonic acid was blocked by La3+, membrane depolarization and SKF 96365, but was unaffected by nitrendipine or BAY K 8644. Furthermore, Ca2+ influx stimulated by BHQ was increased at alkaline pHo and BHQ stimulated the influx of both 45Ca2+ and 133Ba2+ to the same extent. These results demonstrate that the agonist-activated Ca(2+)-influx pathway and the pathway activated by depletion of the agonist-sensitive internal Ca2+ store are indistinguishable.
2,5-Di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a potent inhibitor of liver microsomal ATP-dependent Ca2+ sequestration (Moore, G. A., McConkey, D. J., Kass, G. E. N., O'Brien, P. J., and Orrenius, S. (1987) FEBS Lett. 224, 331-336), produced a concentration-dependent, rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated rat hepatocytes (EC50 = 1-2 microM). The amplitude of the [Ca2+]i increase was essentially identical with that produced by vasopressin, but the tBuBHQ-stimulated [Ca2+]i increase remained sustained for 15-20 min. Vasopressin added 2-3 min after tBuBHQ caused [Ca2+]i to rapidly return to basal levels; however, tBuBHQ added after vasopressin resulted in a Ca2+ transient rather than a sustained [Ca2+]i elevation. Ca2+ influx was not stimulated in tBuBHQ-treated hepatocytes, but was markedly enhanced upon addition of vasopressin. Depletion of the endoplasmic reticular Ca2+ pool by the addition of vasopressin to hepatocytes incubated in low Ca2+ medium virtually abolished the tBuBHQ-mediated [Ca2+]i rise and vice versa. In saponin-permeabilized hepatocytes, tBuBHQ released Ca2+ from the same nonmitochondrial, ATP-dependent Ca2+ pool which was released by inositol 1,4,5-trisphosphate. Furthermore, tBuBHQ-induced Ca2+ release in saponin-permeabilized cells was not inhibited by neomycin, and tBuBHQ did not produce any apparent accumulation of inositol phosphates in intact hepatocytes. The rate of passive efflux of Ca2+ from Ca2+-loaded hepatic microsomes was unaltered by tBuBHQ. Thus, tBuBHQ inhibits ATP-dependent Ca2+ sequestration via a direct effect on the endoplasmic reticulum Ca2+ pump, resulting in net Ca2+ release and elevation of [Ca2+]i. Taken together, our results show that in the absence of hormonal stimuli, excess Ca2+ is only slowly cleared from the hepatocyte cytosol, indicating that the basal rate of Ca2+ removal by the plasma membrane Ca2+ pump and mitochondria is slow. Furthermore, Ca2+-mobilizing hormones appear to stimulate an active process of Ca2+ removal from hepatocyte cytosol which does not depend on re-uptake into the endoplasmic reticulum.
Endothelial cells obtained from human umbilical chord have been studied by the patch clamp method. An ion channel is described that is activated by microM concentrations of histamine and shows a slow run-down in cell-attached patches. After excision, channel activity quickly runs down to zero open probability. In symmetrical potassium concentrations (140 mM K in the bath and the pipette), the single channel conductance is 28 +/- 2 pS and the reversal potential is 0.3 +/- 0.8 mV (mean +/- SEM, n = 4). With 140 mM Na in the pipette, the conductance is 26 +/- 2 pS. A reversal potential of -1.5 +/- 0.9 mV (n = 7) was measured. With 60 mM Ca and 70 mM Na in the pipette, 140 mM K in the bath, the reversal potential was -11 +/- 3 mV, the single channel conductance in 16 +/- 3 pS (n = 5). The single channel conductance in 110 mM Ca (pipette) and 140 mM K (bath) is 8 +/- 2 pS and the reversal potential is -18 +/- 6 mV (n = 3). From analysis of the reversal potentials, a permeation ratio of K:Na:Ca = 1:0.9:0.2 was calculated. This ligand-gated non-selective cation channel in human endothelial cells is Ca permeable and could induce a sustained agonist mediated Ca influx.
Olfactory transduction is thought to be mediated by a G protein-coupled increase in intracellular adenosine 3',5'-monophosphate (cAMP) that triggers the opening of cAMP-gated cation channels and results in depolarization of the plasma membrane of olfactory neurons. In olfactory neurons isolated from the channel catfish, Ictalurus punctatus, stimulation with olfactory stimuli (amino acids) elicits an influx of calcium that leads to a rapid increase in intracellular calcium. In addition, in a reconstitution assay a plasma membrane calcium channel has been identified that is gated by inositol-1,4,5-trisphosphate (IP3), which could mediate this calcium influx. Together with previous studies indicating that stimulation with olfactory stimuli leads to stimulation of phosphoinositide turnover in olfactory cilia, these data suggest that an influx of calcium triggered by odor stimulation of phosphoinositide turnover may be an alternate or additional mechanism of olfactory transduction.
The release of Ca2+ from intracellular stores by sub-optimal doses of inositol trisphosphate has been shown to be dose-related ('quantal'), and a simple model is proposed here to account for this phenomenon. It is suggested that there is a regulatory Ca2(+)-binding site on, or associated with, the luminal domain of the InsP3 receptor, which allosterically controls Ca2+ efflux, and the affinity for Ca2+ of that site is modulated by InsP3 binding to the cytoplasmic domain of the receptor; a similar mechanism applied to the ryanodine receptor might also explain some aspects of Ca2(+)-induced Ca2+ release. The stimulated entry of Ca2+ into a cell which occurs upon activation of inositide-linked receptors has been variously and confusingly proposed to be regulated by InsP3, InsP4, and/or a 'capacitative' Ca2+ pool; the mechanism of InsP3 receptor action suggested here is shown to lead to a potential reconciliation of all these conflicting proposals.
A model is proposed for the mechanism by which activation of surface membrane receptors causes sustained Ca2+ entry into cells from the extracellular space. Reassessment of previously published findings on the behavior of receptor-regulated intracellular Ca2+ pools leads to the conclusion that when such pools are empty, a pathway from the extracellular space to the pool is opened; conversely when the pool is filled, the pathway is closed and it becomes relatively stable to depletion by low Ca2+ media or chelating agents. The biphasic nature of agonist-activated Ca2+-mobilization is thus seen as an initial emptying of the intracellular Ca2+ pool by inositol (1,4,5) trisphosphate, followed by rapid entry of Ca2+ into the pool and, in the continued presence of inositol (1,4,5) trisphosphate, into the cytosol. On withdrawal of agonist, inositol (1,4,5) trisphosphate is then rapidly degraded, the pathway from the pool to the cytosol is closed, and rapid entry from the outside continues until the Ca2+ content of the pool reaches a level that inactivates Ca2+ entry. This capacitative model allows for Ca2+ release and Ca2+ entry to be controlled by a single messenger, inositol (1,4,5) trisphosphate.
Hydrolysis of membrane-associated phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)-P2) to water soluble inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is a common response by many different kinds of cells to a wide variety of external stimuli (see refs 1 and 2 for review). Ins (1,4,5)P3 is a putative second messenger which increases intracellular Ca2+ by mobilizing internal Ca2+ stores, a hypothesis which has been substantiated by studies with chemically permeabilized cells and with isolated microsomal membrane fractions. But the possibility that Ins(1,4,5)P3 could induce in intact cells an influx of external Ca2+ through transmembrane channels, originally hypothesized by Michell in 1975, has never been directly tested. We report here single-channel recordings of an Ins(1,4,5)P3-activated conductance in excised patches of T-lymphocyte plasma membrane. The Ins(1,4,5)P3-activated transmembrane channel appears to be identical to the recently described mitogen-regulated, voltage-insensitive Ca2+ permeable channel involved in T-cell activation. We suggest that Ins(1,4,5)P3 acts as the second messenger mediating transmembrane Ca2+ influx through specific Ca2+-permeable channels in mitogen-stimulated T-cell activation.
Two complementary experimental methods have been used to examine mitogen-induced transmembrane conductances in human B cells using the Daudi cell line as a model for human B cell activation. Spectrofluorometry was used to investigate mitogen-induced changes in [Ca++]i and transmembrane potential. Activation of human B cells with anti-mu antibodies resulted in a biphasic rise in [Ca++]i, the second phase being mediated by the influx of extracellular Ca++. Ca++ influx was inhibited by high [K+]e, suggesting that this influx was transmembrane potential sensitive. Membrane currents of Daudi cells were investigated using voltage clamp techniques. Before mitogenic stimulation, the cells were electrically quiet. Within several minutes of the addition of anti-mu antibodies to the bath solution, inward currents were observed at negative voltages. Whole-cell currents changed instantly with voltage steps and were transmembrane potential sensitive in that at potentials more positive than -40 mV no currents were detectable. A similar conductance was also activated by the introduction of IP3 into the intracellular solution, suggesting that IP3 generation after surface IgM crosslinking is involved in the activation of this conductance. Both anti-mu and IP3 induced currents were blocked by 1 mM La , which is known to block Ca++ channels. These results strongly support the presence of membrane Ca++ channels in human B cells that function in the early stages of activation. Changes in transmembrane potential appear to be important in regulating Ca++ influx. These mechanisms work in concert to regulate the level of [Ca++]i during the early phases of human B cell activation.