Fig 5 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
4−AP does not affect closed−state inactivation in Kv2.1/Kv9.3 heterotetramers. (A−B) Representative current recordings under control (upper) and 4−AP (bottom) conditions elicited by the pulse protocols shown on top. The pulse protocols were used to investigate the induction (A) and recovery (B) of closed−state inactivation. The dotted lines represent the exponential fit to determine these rate constants. (C−D) The normalized plot of induction at −45 mV (C) and recovery at −100 mV (D) of closed−state inactivation was obtained as described in Fig 4. Solid lines represent the exponential fit. 4−AP decreased
Source publication
The voltage-gated K+ (Kv) channel subunits Kv2.1 and Kv2.2 are expressed in almost every tissue. The diversity of Kv2 current is increased by interacting with the electrically silent Kv (KvS) subunits Kv5-Kv6 and Kv8-Kv9, into functional heterotetrameric Kv2/KvS channels. These Kv2/KvS channels possess unique biophysical properties and display a mo...
Similar publications
Background/aims:
Early repolarization syndrome (ERS) has been recently recognized as early repolarization pattern with idiopathic ventricular fibrillation. However, the genetic background of ERS has not been fully understood.
Methods:
A Chinese family with sudden cardiac death associated with ERS was investigated. Direct sequencing of ERS suscep...
Citations
... The S1-S4 region represents the voltage-sensing domain, and the cytoplasmic N-and C-termini are relevant for subunit assembly, trafficking and functional channel regulation through signalling cascades. Notably, it is well established that KVS affect Kv2 currents by altering membrane trafficking, but also by modifying the voltage dependence of activation and inactivation as well as the gating properties (Bocksteins, 2016;Bocksteins & Snyders, 2012;Stas et al, 2015). ...
Voltage-gated K ⁺ (K V ) channels govern K+-ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent K V channels (K V S), however, are unable to conduct currents on their own. It has been assumed that these K V S must obligatorily assemble with subunits from the K V 2 family into heterotetrameric channels, thereby giving raise to currents distinct from those of homomeric K V 2 channels.
Herein, we show that K V S subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant K V 7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of K V S and K V 7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this K V S and K V 7 interaction may occur. Our finding demonstrate that K V cross-family interaction is much more versatile than previously thought – possibly serving nature to shape potassium conductance to the needs of individual cell types.
... For example, Kv6.4 alters the voltage-dependence of Kv2.1 subunits , and alters dynamics of the S6 pore-gating apparatus (Pisupati et al., 2018). Notably, the S6 helical bundle crossing of Kv2.1 constricts asymmetrically during channel inactivation in ways that are allosterically modulated by 4-aminopyridine (Fernández-Mariño et al., 2023), and KvS channels allosterically alter interactions with 4-aminopyridine (Stas et al., 2015). These observations make it seem likely that KvS subunits could allosterically disrupt RY785 inhibition. ...
... ;https://doi.org/10.1101https://doi.org/10. /2024 to RY785 may vary among KvS subunits considering this is likely with other central cavity drugs such as tetraethylammonium and 4-aminopyridine (Post et al., 1996;Thorneloe and Nelson, 2003;Stas et al., 2015). While Kv8.1/Kv2.1 channels appeared to be homogenously susceptible to RY785 (with Hill slope close to 1), other KvS subunits might present multiple pharmacologically-distinct heteromer populations. ...
... Third, concentrations of RY785 which partially blocked Kv8.1/Kv2.1 modified the gating of the voltage-activated conductance, indicating that RY785 can create a conductance with novel kinetics, or potentially even a gain-of-function under certain conditions. Notably, 4-aminopyridine can increase Kv2 or KvS conductance under specialized conditions (Stas et al., 2015). As discussed above, channel statedependent binding is expected to influence the affinity of RY785. ...
KvS proteins are pore-forming voltage-gated potassium channel subunits that must co-assemble into heterotetramers with Kv2.1 (KCNB1) or Kv2.2 (KCNB2) subunits to form functional channels. In mammals, KvS subunits encompass the Kv5.1 (KCNF1), Kv6.1 (KCNG1), Kv6.2 (KCNG2), Kv6.3 (KCNG3), Kv6.4 (KCNG4), Kv8.1 (KCNV1), Kv8.2 (KCNV2), Kv9.1 (KCNS1), Kv9.2 (KCNS2), and Kv9.3 (KCNS3) proteins. While Kv2 proteins are broadly expressed in electrically excitable cells, KvS mRNAs are enriched in unique subsets of these cells. The physiological functions of KvS-containing channels are poorly understood and no drugs are known to selectively modulate KvS subunits. Here, we identify a pair of potent Kv2-selective inhibitors which distinguish conductances of KvS-containing channels. We find that conductances of KvS-containing channels are resistant to the pore-blocker RY785 yet remain sensitive to the voltage sensor modulator guangxitoxin-1E (GxTX). We show that this pattern of inhibitor sensitivities is consistent among subunits from Kv5, Kv6, Kv8, and Kv9 families. By deploying these inhibitors we find that mouse superior cervical ganglion neurons have conductances consistent with Kv2, but not KvS-containing channels. In contrast, mouse and human dorsal root ganglion neurons have conductances consistent with KvS/Kv2 heteromeric channels. These results provide an approach to pharmacologically distinguish KvS-containing from Kv2-only channels, and identify endogenous KvS conductances.
... Plasmids encoding untagged and GFP-, DsRed-or HA-tagged Kv2.1 and Kv2.2 have been previously described (Lim et al., 2000;Bishop et al., 2018;Kirmiz et al., 2018a). Plasmid encoding human Kv5.1 was generously provided by Dr. Dirk Snyders (Stas et al., 2015), and a plasmid encoding human Kv5.1 with a C-terminal GFP tag was generated by Genscript. The Kv5.1BBS construct was generated at Genscript by inserting a bungarotoxin-binding site (GGWRYYESSLLPYPDGG) at amino acid 215 in the external S1-S2 loop of Kv5.1, as well as an HA tag at the C-terminus. ...
... ;https://doi.org/10.1101https://doi.org/10. /2024 Notably, KvS subunits also modify the pharmacological response of Kv2/KvS heteromers to another pore blocker, 4-aminopyridine (Stas et al., 2015). Another key function of Kv2 channels is to organize ER-PM junctions on neuronal somata and proximal dendrites (Johnson et al., 2019;Vierra and Trimmer, 2022). ...
Voltage-gated K+ channels of the Kv2 family are highly expressed in brain and play dual roles in regulating neuronal excitability and in organizing endoplasmic reticulum - plasma membrane (ER-PM) junctions. Studies in heterologous cells suggest that the two pore forming alpha subunits Kv2.1 and Kv2.2 assemble with electrically silent KvS subunits to form heterotetrameric channels with distinct biophysical properties. Here, using mass spectrometry-based proteomics, we identified five KvS subunits as components of native Kv2.1 channels immunopurified from mouse brain, the most abundant being Kv5.1. We found that Kv5.1 co-immunoprecipitates with Kv2.1 and to a lesser extent with Kv2.2 from brain lysates, and that Kv5.1 protein levels are decreased by 70% in Kv2.1 knockout mice and 95% in Kv2.1/2.2 double knockout mice. Multiplex immunofluorescent labelling of rodent brain sections revealed that in neocortex Kv5.1 immunolabeling is apparent in a large percentage of Kv2.1 and Kv2.2-positive layer 2/3 neurons, and in a smaller percentage of layer 5 and 6 neurons. At the subcellular level, Kv5.1 is co-clustered with Kv2.1 and Kv2.2 at ER-PM junctions in cortical neurons, although clustering of Kv5.1-containing channels is reduced relative to homomeric Kv2 channels. We also found that in heterologous cells coexpression with Kv5.1 reduces the clustering and alters the pharmacological properties of Kv2.1 channels. Together, these findings demonstrate that the Kv5.1 electrically silent subunit is a component of a substantial fraction of native brain Kv2 channels, and that its incorporation into heteromeric channels can impact diverse aspects of Kv2 channel function.
... In addition, recent studies have shown that 4-AP blocks voltage-sensitive K + channels, specifically those that contain the Kv2.1 and Kv2.2 α-subunits, probably because 4-AP binds to the central part of the channel (Muñoz-Caro and Niño, 2002;Stas et al., 2015;Page et al., 2018); these K + channels are expressed in almost all tissues, and the current of these subunits increases when interacting with the Kv5, Kv8 and Kv9 subunits, which are electrically silent (Bocksteins, 2016). Consequently, the interaction of the Kv2.1 and Kv2.2 α-subunits with the Kv5, Kv8 and Kv9 subunits, makes them a more desirable pharmacological and therapeutic target due to their unique biophysical properties. ...
... Consequently, the interaction of the Kv2.1 and Kv2.2 α-subunits with the Kv5, Kv8 and Kv9 subunits, makes them a more desirable pharmacological and therapeutic target due to their unique biophysical properties. In addition, 4-AP, in a single dose of 10 mg in mouse and human tissues, potentiated Kv6.4 currents in the K + channels (Bocksteins et al., 2014;Stas et al., 2015;Taranto-Montemurro et al., 2017). ...
Aminopyridines constitute a drug family with the ability to enhance synaptic transmission. In particular, 4‑aminopyridine (4‑AP) has been used as a model of generalized seizures. 4‑AP is a K+ channel blocker, but its mechanism of action has not yet been fully described; some evidence has shown that it acts on the K+ channel types Kv1.1, Kv1.2, Kv1.4 and Kv4, which are localized in the axonic terminals of pyramidal neurons and interneurons. When 4‑AP blocks the K+ channels it triggers depolarization and prolongs the action potential in the neuron, which causes nonspecific neurotransmitter release. Among these neurotransmitters, glutamate is the principal excitatory neurotransmitter released in the hippocampus. Once glutamate is released, it reaches its ionotropic and metabotropic receptors continuing the neuronal depolarization chain and propagation of hyperexcitability. This brief review is focused on the use of 4‑AP as an effective seizure model for testing antiseizure drugs in relevant in vitro and in vivo studies.
... We observed that Htr4 mRNA levels recovered after 5-AZA treatment in parallel with pain relief, which indicates that Htr4 may produce antinociceptive effects as an inhibitory receptor subtype of 5-HT in the lumbar spinal cord after nerve injury. Moreover, KCNF1 is a potassium channel gene and encodes Kv5.1, which can form a functional complex with Kv2.1, resulting in physiological effects [67,68]. The contribution of the Kv5.1 channel to the regulation of NPP has not been investigated, and our results first showed that the decrease in Kv5.1 mRNA was reversed after 5-AZA treatment along with pain relief in CCI/SNL rats, suggesting that Kv5.1 may be involved in the occurrence and development of NPP. ...
To investigate the role of DNA methylation in modulating chronic neuropathic pain (NPP), identify possible target genes of DNA methylation involved in this process, and preliminarily confirm the medicinal value of the DNA methyltransferases (DNMTs) inhibitor 5-azacytidine (5-AZA) in NPP by targeting gene methylation. Two rat NPP models, chronic constriction injury (CCI) and spinal nerve ligation (SNL), were used. The DNA methylation profiles in the lumbar spinal cord were assayed using an Arraystar Rat RefSeq Promoter Array. The underlying genes with differential methylation were then identified and submitted to Gene Ontology and pathway analysis. Methyl-DNA immunoprecipitation quantitative PCR (MeDIP-qPCR) and quantitative reverse transcription-PCR (RT-qPCR) were used to confirm gene methylation and expression. The protective function of 5-AZA in NPP and gene expression were evaluated via behavioral assays and RT-qPCR, respectively. Analysis of the DNA methylation patterns in the lumbar spinal cord indicated that 1205 differentially methylated fragments in CCI rats were located within DNA promoter regions, including 638 hypermethylated fragments and 567 hypomethylated fragments. The methylation levels of Grm4, Htr4, Adrb2, Kcnf1, Gad2, and Pparg, which are associated with long-term potentiation (LTP) and glutamatergic synapse pathways, were increased with a corresponding decrease in their mRNA expression, in the spinal cords of CCI rats. Moreover, we found that the intraperitoneal injection of 5-AZA (4 mg/kg) attenuated CCI- or SNL-induced mechanical allodynia and thermal hyperalgesia. Finally, the mRNA expression of hypermethylated genes such as Grm4, Htr4, Adrb2, Kcnf1, and Gad2 was reversed after 5-AZA treatment. CCI induced widespread methylation changes in the DNA promoter regions in the lumbar spinal cord. Intraperitoneal 5-AZA alleviated hyperalgesia in CCI and SNL rats, an effect accompanied by the reversed expression of hypermethylated genes. Thus, DNA methylation inhibition represents a promising epigenetic strategy for protection against chronic NPP following nerve injury. Our study lays a theoretical foundation for 5-AZA to become a clinical targeted drug.
... Similar to other KvS subfamilies, KCNF1 is unable to form functional homotetramers, but is able to form heterotetramers with Kv2.1, whereby modulating its electrophysiological properties [32,33]. The function of KCNF1 in cell proliferation and cancer progression has not been previously explored. ...
Lung cancer continues to be the leading cause of cancer death in the United States. Despite recent advances, the five-year survival rate for lung cancer compared to other cancers still remains fairly low. The discovery of molecular targets for lung cancer is key to the development of new approaches and therapies. Electrically silent voltage-gated potassium channel (KvS) subfamilies, which are unable to form functional homotetramers, are implicated in cell-cycle progression, cell proliferation and tumorigenesis. Here, we analyzed the expression of KvS subfamilies in human lung tumors and identified that potassium voltage-gated channel subfamily F member 1 (KCNF1) was up-regulated in non-small cell lung cancer (NSCLC). Silencing of KCNF1 in NSCLC cell lines reduced cell proliferation and tumor progression in mouse xenografts, re-established the integrity of the basement membrane, and enhanced cisplatin sensitivity. KCNF1 was predominately localized in the nucleoplasm and likely mediated its functions in an ion-independent manner. We identified integrin β4 subunit (ITGB4) as a downstream target for KCNF1. Our findings suggest that KCNF1 promotes lung cancer by enhancing ITGB4 signaling and implicate KCNF1 as a novel therapeutic target for lung cancer.
... For example, the pore blocker 4-aminopyridine requires opening of the S6 gate of Shaker Kv1 channels to block and then stabilizes pores in closed conformations (Armstrong and Loboda, 2001). Interestingly, 4-aminopyridine can cause an increase in the conductance of Kv6.4/ Kv2.1 heteromeric channels (Stas et al., 2015). The Kv2 blocker RY785 accelerates channel deactivation to trap itself (Marquis and Sack, 2022). ...
A drug that blocks the cardiac myocyte voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene (hERG) carries a potential risk of long QT syndrome and life-threatening cardiac arrhythmia, including Torsade de Points Interestingly, certain hERG blockers can also facilitate hERG activation to increase hERG currents, which may reduce proarrhythmic potential. However, the molecular mechanism involved in the facilitation effect of hERG blockers remains unclear. The hallmark feature of the facilitation effect by hERG blockers is that a depolarizing preconditioning pulse shifts voltage-dependence of hERG activation to more negative voltages. Here we utilize a D540K hERG mutant to study the mechanism of the facilitation effect. D540K hERG is activated by not only depolarization but also hyperpolarization. This unusual gating property enables tests of the mechanism by which voltage induces facilitation of hERG by blockers. With D540K hERG, we find that nifekalant, a hERG blocker and Class III antiarrhythmic agent, blocks and facilitates not only current activation by depolarization but also current activation by hyperpolarization, suggesting a shared gating process upon depolarization and hyperpolarization. Moreover, in response to hyperpolarizing conditioning pulses, nifekalant facilitates D540K hERG currents but not wild-type currents. Our results indicate that induction of facilitation is coupled to pore opening, not voltage per se We propose that gated access to the hERG central cavity underlies the voltage-dependence of induction of facilitation. This study identifies hERG channel pore gate opening as the conformational change facilitated by nifekalant, a clinically important antiarrhythmic agent. Significance Statement Nifekalant is a clinically important antiarrhythmic agent and a hERG blocker which can also facilitate voltage-dependent activation of hERG channels after a preconditioning pulse. Here we show that the mechanism of action of the preconditioning pulse is to open a conductance gate to enable drug access to a facilitation site. Moreover, we find that facilitation increases hERG currents by altering pore dynamics, rather than acting through voltage sensors.
... This makes them potential therapeutic targets, especially since it was demonstrated that Kv2.1/Kv6.4 channels are modulated differently by the well-characterized drug 4-aminopyridine compared to Kv2.1 homotetramers and other Kv2/KvS heterotetramers (40). It has been shown and confirmed by our TEA experiments (Fig. 8) that the pharmacological characteristics of heteromeric Kv channel complexes are affected by the stoichiometry of the subunits involved (23,48,49). ...
... heterotetramers with biophysical properties that differed clearly from those of Kv2.1 homotetramers, and the obtained kinetics were comparable to what has been reported before(Table 1)(30,(35)(36)(37)(38)(39)(40). The most significant difference with Kv2.1 homotetramers is an approximately −40-mV shift in the voltage dependence of inactivation and an activation curve with a shallower slope factor k (Table 1). ...
Significance
Voltage-gated potassium (Kv) channels play a key role in cellular electrical excitability. While various Kv subunits assemble to homotetrameric functional channels, the silent subfamilies KvS exclusively form heterotetramers with Kv2 subunits and thus regulate their biophysical properties in a tissue-specific way. Despite the vast functional research, key aspects of the heterotetrameric architecture remain controversial, including the stoichiometry with which KvS and Kv2 can assemble. We used concatemers and single subunit counting in combination to show that Kv2/Kv6 assemble in a 2:2 stoichiometry following the general dimer-of-dimer mechanism for channel formation. We demonstrate how to objectively choose the most likely model for single subunit counting data, which is applicable for any multimeric complex and will help choosing models confidently.
... Each Kv subunit contains six transmembrane segments; 1 to 4 form the voltage-sensing domain and segments 5 and 6 form the central pore structure. Activation of Kv channels is thought to be regulated by dynamic coupling of the cytoplasmic linker between transmembrane segments 4 and 5 and the lower half of segment 6 [122]. Interestingly, KCNG4 p.Arg365His is located in the regulatory linker between transmembrane segments 4 and 5, and p.Arg474His in the cytoplasmic C-terminal domain, 13 amino acids from transmembrane segment 6. ...
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described. We performed whole-exome sequencing analysis in 132 patients from 34 multi-incident families, which nominated likely pathogenic variants for MS in 12 genes of the innate immune system that regulate the transcription and activation of inflammatory mediators. Rare missense or nonsense variants were identified in genes of the fibrinolysis and complement pathways (PLAU, MASP1, C2), inflammasome assembly (NLRP12), Wnt signaling (UBR2, CTNNA3, NFATC2, RNF213), nuclear receptor complexes (NCOA3), and cation channels and exchangers (KCNG4, SLC24A6, SLC8B1). These genes suggest a disruption of interconnected immunological and pro-inflammatory pathways as the initial event in the pathophysiology of familial MS, and provide the molecular and biological rationale for the chronic inflammation, demyelination and neurodegeneration observed in MS patients.
... Earlier, it was shown that currents through K v 2.1 channels that are normally inhibited by 4-AP were potentiated by the substance, but only when K v 6.4 subunits were co-expressed (Stas et al., 2015). As K v 2.1 and K v 6.4 co-assemble into functional channels (reviewed in Bocksteins, 2016), this suggested that K v 6.4 largely determined altered 4-AP sensitivity of the resulting heteromeric channels. ...
... As K v 2.1 and K v 6.4 co-assemble into functional channels (reviewed in Bocksteins, 2016), this suggested that K v 6.4 largely determined altered 4-AP sensitivity of the resulting heteromeric channels. 4-AP suppressed closed state inactivation of K v 2.1/K v 6.4 resulting in exclusive current potentiation of currents through those heteromers (Stas et al., 2015). In contrast, K v 12.1 channels did not inactivate in our experiments, and thus 4-AP probably does not potentiate K v 12.1-mediated currents through a similar mechanism. ...
The three members of the ether-à-go-go-gene-like (Elk; Kv12.1-Kv12.3) family of voltage-gated K+ channels are predominantly expressed in neurons, but only little information is available on their physiological relevance. It was shown that Kv12.2 channels modulate excitability of hippocampal neurons, but no native current could be attributed to Kv12.1 and Kv12.3 subunits yet. This may appear somewhat surprising, given high expression of their mRNA transcripts in several brain areas. Native Kv12 currents may have been overlooked so far due to limited knowledge on their biophysical properties and lack of specific pharmacology. Except for Kv12.2, appropriate genetically modified mouse models have not been described; therefore, identification of Kv12-mediated currents in native cell types must rely on characterization of unique properties of the channels. We focused on recombinant human Kv12.1 to identify distinct properties of these channels. We found that Kv12.1 channels exhibited significant mode shift of activation, i.e., stabilization of the voltage sensor domain in a “relaxed” open state after prolonged channel activation. This mode shift manifested by a slowing of deactivation and, most prominently, a significant shift of voltage dependence to hyperpolarized potentials. In contrast to related Kv11.1, mode shift was not sensitive to extracellular Na+, which allowed for discrimination between these isoforms. Sensitivity of Kv12.1 and Kv11.1 to the broad-spectrum K+ antagonist 4-aminopyridine was similar. However, 4-AP strongly activated Kv12.1 channels, but it was an inhibitor of Kv11 channels. Interestingly, the agonist of Kv11 channels NS1643 also differentially modulated the activity of these channels, i.e., NS1643 activated Kv11.1, but strongly inhibited Kv12.1 channels. Thus, these closely related channels are distinguished by inverse pharmacological profiles. In summary, we identified unique biophysical and pharmacological properties of Kv12.1 channels and established straightforward experimental protocols to characterize Kv12.1-mediated currents. Identification of currents in native cell types with mode shift that are activated through 4-AP and inhibited by NS1643 can provide strong evidence for contribution of Kv12.1 to whole cell currents.
