A self-inactivating invertebrate opsin optically drives biased signaling toward Gßγ-dependent ion channel modulation.

Animal opsins, light-sensitive G protein-coupled receptors, have been used for optogenetic tools to control G protein-dependent signaling pathways. Upon G protein activation, the Gα and Gßγ subunits drive different intracellular signaling pathways, leading to complex cellular responses. For some purposes, Gα- and Gßγ-dependent signaling needs to be separately modulated, but these responses are simultaneously evoked due to the 1:1 stoichiometry of Gα and Gßγ Nevertheless, we show temporal activation of G protein using a self-inactivating invertebrate opsin, Platynereis c-opsin1, drives biased signaling for Gßγ-dependent GIRK channel activation in a light-dependent manner by utilizing the kinetic difference between Gßγ-dependent and Gα-dependent responses. The opsin-induced transient Gi/o activation preferentially causes activation of the kinetically fast Gßγ-dependent GIRK channels rather than slower Gi/oα-dependent adenylyl cyclase inhibition. Although similar Gßγ-biased signaling properties were observed in a self-inactivating vertebrate visual pigment, Platynereis c-opsin1 requires fewer retinal molecules to evoke cellular responses. Furthermore, the Gßγ-biased signaling properties of Platynereis c-opsin1 are enhanced by genetically fusing with RGS8 protein, which accelerates G protein inactivation. The self-inactivating invertebrate opsin and its RGS8-fusion protein can function as optical control tools biased for Gßγ-dependent ion channel modulation.

Conformational rearrangements in the second voltage sensor domain switch PIP2- and voltage-gating modes in two-pore channels.

Two-pore channels (TPCs) are activated by phosphatidylinositol bisphosphate (PIP2) binding to domain I and/or by voltage sensing in domain II (DII). Little is known about how these two stimuli are integrated, and how each TPC subtype achieves its unique preference. Here, we show that distinct conformations of DII-S4 in the voltage-sensor domain determine the two gating modes. DII-S4 adopts an intermediate conformation, and forced stabilization in this conformation was found to result in a high PIP2-dependence in primarily voltage-dependent TPC3. In TPC2, which is PIP2-gated and nonvoltage-dependent, a stabilized intermediate conformation does not affect the PIP2-gated currents. These results indicate that the intermediate state represents the PIP2-gating mode, which is distinct from the voltage-gating mode in TPCs. We also found in TPC2 that the tricyclic antidepressant desipramine induces DII-S4-based voltage dependence and that naringenin, a flavonoid, biases the mode preference from PIP2-gating to desipramine-induced voltage gating. Taken together, our study on TPCs revealed an unprecedented mode-switching mechanism involving conformational changes in DII-S4, and its active role in integrating voltage and PIP2 stimuli.

Structural determinants of the direct inhibition of GIRK channels by Sigma-1 receptor antagonist.

G-protein-gated inward rectifier K+ (GIRK) channels play a critical role in the regulation of the excitability of cardiomyocytes and neurons and include GIRK1, GIRK2, GIRK3 and GIRK4 subfamily members. BD1047 dihydrobromide (BD1047) is one of the representative antagonists of the multifunctional Sigma-1 receptor (S1R). In the analysis of the effect of BD1047 on the regulation of Gi-coupled receptors by S1R using GIRK channel as an effector, we observed that BD1047, as well as BD1063, directly inhibited GIRK currents even in the absence of S1R and in a voltage-independent manner. Thus, we aimed to clarify the effect of BD1047 on GIRK channels and identify the structural determinants. By electrophysiological recordings in Xenopus oocytes, we observed that BD1047 directly inhibited GIRK channel currents, producing a much stronger inhibition of GIRK4 compared to GIRK2. It also inhibited ACh-induced native GIRK current in isolated rat atrial myocytes. Chimeric and mutagenesis studies of GIRK2 and GIRK4 combined with molecular docking analysis demonstrated the importance of Leu77 and Leu84 within the cytoplasmic, proximal N-terminal region and Glu147 within the pore-forming region of GIRK4 for inhibition by BD1047. The activator of GIRK channels, ivermectin, competed with BD1047 at Leu77 on GIRK4. This study provides us with a novel inhibitor of GIRK channels and information for developing pharmacological treatments for GIRK4-associated diseases.

A novel ion conducting route besides the central pore in an inherited mutant of G-protein-gated inwardly rectifying K+ channel.

G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important physiological roles in various organs. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited GIRK mutants. By the two-electrode voltage-clamp analysis of GIRK mutants heterologously expressed in Xenopus oocytes, we observed that Kir3.2 G156S permeates Li+ better than Rb+ , while T154del or L173R of Kir3.2 and T158A of Kir3.4 permeate Rb+ better than Li+ , suggesting a unique conformational change in the G156S mutant. Applications of blockers of the selectivity filter (SF) pathway, Ba2+ or Tertiapin-Q (TPN-Q), remarkably increased the Li+ -selectivity of Kir3.2 G156S but did not alter those of the other mutants. In single-channel recordings of Kir3.2 G156S expressed in mouse fibroblasts, two types of events were observed, one attributable to a TPN-Q-sensitive K+ current and the second a TPN-Q-resistant Li+ current. The results show that a novel Li+ -permeable and blocker-resistant pathway exists in G156S in addition to the SF pathway. Mutations in the pore helix, S148F and T151A also induced high Li+ permeation. Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations. KEY POINTS: G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important roles in controlling excitation of cells in various organs, such as the brain and the heart. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited mutants of Kir3.2 and Kir3.4. Here we show that a novel Na+ , Li+ -permeable and blocker-resistant pathway exists in an inherited mutant, Kir3.2 G156S, in addition to the conventional ion conducting pathway formed by the selectivity filter (SF). Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations.

Phosphoinositide regulates dynamic movement of the S4 voltage sensor in the second repeat in two-pore channel 3.

The two-pore channels (TPCs) are voltage-gated cation channels consisting of single polypeptides with two repeats of a canonical 6-transmembrane unit. TPCs are known to be regulated by various physiological signals such as membrane voltage and phosphoinositide (PI). The fourth helix in the second repeat (second S4) plays a major role in detecting membrane voltage, whereas the first repeat contains a PI binding site. Therefore, each of these stimuli is detected by a unique repeat to regulate the gating of the TPC central pore. How these various stimuli regulate the dynamic structural rearrangement of the TPC molecule remain unknown. Here, we found that PI binding to the first repeat in TPC3 regulates the movement of the distally located second S4 helix, showing that the PI-binding signal is not confined to the pore gate but also transmitted to the voltage sensor. Using voltage clamp fluorometry, measurement of gating charges, and Cys-accessibility analysis, we observed that PI binding significantly potentiates the voltage dependence of the movement of the second S4 helix. Notably, voltage clamp fluorometry analysis revealed that the voltage-dependent movement of the second S4 helix occurred in two phases, of which the second phase corresponds to the transfer of the gating charges. This movement was observed in the voltage range where gate-opening occurs and was potentiated by PI. In conclusion, this regulation of the second S4 helix by PI indicates a tight inter-repeat coupling within TPC3, a feature which might be conserved among TPC family members to integrate various physiological signals.

AMPA glutamate receptors are required for sensory-organ formation and morphogenesis in the basal chordate.

AMPA-type glutamate receptors (GluAs) mediate fast excitatory transmission in the vertebrate central nervous system (CNS), and their function has been extensively studied in the mature mammalian brain. However, GluA expression begins very early in developing embryos, suggesting that they may also have unidentified developmental roles. Here, we identify developmental roles for GluAs in the ascidian Ciona intestinalis Mammals express Ca2+-permeable GluAs (Ca-P GluAs) and Ca2+-impermeable GluAs (Ca-I GluAs) by combining subunits derived from four genes. In contrast, ascidians have a single gluA gene. Taking advantage of the simple genomic GluA organization in ascidians, we knocked down (KD) GluAs in Ciona and observed severe impairments in formation of the ocellus, a photoreceptive organ used during the swimming stage, and in resorption of the tail and body axis rotation during metamorphosis to the adult stage. These defects could be rescued by injection of KD-resistant GluAs. GluA KD phenotypes could also be reproduced by expressing a GluA mutant that dominantly inhibits glutamate-evoked currents. These results suggest that, in addition to their role in synaptic communication in mature animals, GluAs also have critical developmental functions.

Structural properties determining low K+ affinity of the selectivity filter in the TWIK1 K+ channel.

Canonical K+ channels are tetrameric and highly K+-selective, whereas two-pore-domain K+ (K2P) channels form dimers, but with a similar pore architecture. A two-pore-domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na+ and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange-induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K+-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K+ ions, WT and both variants exhibit an amide-I band at 1680 cm-1 This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K+, a feature very similar to that of the canonical K+ channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K+ affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm-1 band upon changes from K+ to Rb+ or Cs+ conditions. High-speed atomic force microscopy disclosed that TWIK1's surface morphology largely does not change in K+ and Na+ solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful "molecular fingerprints" for assessing the properties of other K+ channels.

Ivermectin and its target molecules: shared and unique modulation mechanisms of ion channels and receptors by ivermectin.

Ivermectin (IVM) is an antiparasitic drug that is used worldwide and rescues hundreds of millions of people from onchocerciasis and lymphatic filariasis. It was discovered by Satoshi Omura and William C. Campbell, to whom the 2015 Nobel Prize in Physiology or Medicine was awarded. It kills parasites by activating glutamate-gated Cl- channels, and it also targets several ligand-gated ion channels and receptors, including Cys-loop receptors, P2X4 receptors and fernesoid X receptors. Recently, we found that IVM also activates a novel target, the G-protein-gated inwardly rectifying K+ channel, and also identified the structural determinant for the activation. In this review, we aim to provide an update and summary of recent progress in the identification of IVM targets, as well as their modulation mechanisms, through molecular structures, chimeras and site-directed mutagenesis, and molecular docking and modelling studies.

Two mutations at different positions in the CNBH domain of the hERG channel accelerate deactivation and impair the interaction with the EAG domain.

KEY POINTS: In the human ether-a-go-go related gene (hERG) channel, both the ether-a-go-go (EAG) domain in the N-terminal and the cyclic nucleotide (CN) binding homology (CNBH) domain in the C-terminal cytoplasmic region are known to contribute to the characteristic slow deactivation. Mutations of Phe860 in the CNBH domain, reported to fill the CN binding pocket, accelerate the deactivation and decrease the fluorescence resonance energy transfer (FRET) efficiencies between the EAG and CNBH domains. An electrostatic interaction between Arg696 and Asp727 in the C-linker domain, critical for HCN and CNG channels, is not formed in the hERG channel. Mutations of newly identified electrostatically interacting pair, Asp727 in the C-linker and Arg752 in the CNBH domains, accelerate the deactivation and decrease FRET efficiency. Voltage-dependent changes in FRET efficiency were not detected. These results suggest that the acceleration of the deactivation by mutations of C-terminal domains is a result of the lack of interaction between the EAG and CNBH domains. ABSTRACT: The human ether-a-go-go related gene (hERG) channel shows characteristic slow deactivation, and the contribution of both of the N-terminal cytoplasmic ether-a-go-go (EAG) domain and the C-terminal cytoplasmic cyclic nucleotide (CN) binding homology (CNBH) domain is well known. The interaction between these domains is known to be critical for slow deactivation. We analysed the effects of mutations in the CNBH domain and its upstream C-linker domain on slow deactivation and the interaction between the EAG and CNBH domains by electrophysiological and fluorescence resonance energy transfer (FRET) analyses using Xenopus oocyte and HEK293T cell expression systems. We first observed that mutations of Phe860 in the CNBH domain, which is reported to fill the CN binding pocket as an intrinsic ligand, accelerate deactivation and eliminate the inter-domain interaction. Next, we observed that the salt bridge between Arg696 and Asp727 in the C-linker domain, which is reported to be critical for the function of CN-regulated channels, is not formed. We newly identified an electrostatically interacting pair critical for slow deactivation: Asp727 and Arg752 in the CNBH domain. Their mutations also impaired the inter-domain interaction. Taking these results together, both mutations of the intrinsic ligand (Phe860) and a newly identified salt bridge pair (Asp727 and Arg752) in the hERG channel accelerated deactivation and also decreased the interaction between the EAG and CNBH domains. Voltage-dependent changes in FRET efficiency between the two domains were not detected. The results suggest that the CNBH domain contributes to slow deactivation of the hERG channel by a mechanism involving the EAG domain.

A ciliary opsin in the brain of a marine annelid zooplankton is ultraviolet-sensitive, and the sensitivity is tuned by a single amino acid residue.

Ciliary opsins were classically thought to function only in vertebrates for vision, but they have also been identified recently in invertebrates for non-visual photoreception. Larvae of the annelid Platynereis dumerilii are used as a zooplankton model, and this zooplankton species possesses a "vertebrate-type" ciliary opsin (named c-opsin) in the brain. Platynereis c-opsin is suggested to relay light signals for melatonin production and circadian behaviors. Thus, the spectral and biochemical characteristics of this c-opsin would be directly related to non-visual photoreception in this zooplankton model. Here we demonstrate that the c-opsin can sense UV to activate intracellular signaling cascades and that it can directly bind exogenous all-trans-retinal. These results suggest that this c-opsin regulates circadian signaling in a UV-dependent manner and that it does not require a supply of 11-cis-retinal for photoreception. Avoidance of damaging UV irradiation is a major cause of large-scale daily zooplankton movement, and the observed capability of the c-opsin to transmit UV signals and bind all-trans-retinal is ideally suited for sensing UV radiation in the brain, which presumably lacks enzymes producing 11-cis-retinal. Mutagenesis analyses indicated that a unique amino acid residue (Lys-94) is responsible for c-opsin-mediated UV sensing in the Platynereis brain. We therefore propose that acquisition of the lysine residue in the c-opsin would be a critical event in the evolution of Platynereis to enable detection of ambient UV light. In summary, our findings indicate that the c-opsin possesses spectral and biochemical properties suitable for UV sensing by the zooplankton model.

Ivermectin activates GIRK channels in a PIP2 -dependent, Gßγ -independent manner and an amino acid residue at the slide helix governs the activation.

KEY POINTS: Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channel in parasites. It is known that IVM binds to the transmembrane domains (TMs) of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. We found that the G-protein-gated inwardly rectifying K+ (GIRK) channel, especially GIRK2, is activated by IVM directly in a Gßγ -independent manner, but the activation is dependent on phosphatidylinositol-4,5-biphosphate (PIP2 ). We identified a critical amino acid residue of GIRK2 for activation by IVM, Ile82, located in the slide helix between the TM1 and the N-terminal cytoplasmic tail domain (CTD). The results demonstrate that the TM-CTD interface in GIRK channel, rather than the TMs, governs IVM-mediated activation and provide us with novel insights on the mode of action of IVM in ion channels. ABSTRACT: Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channel in parasites. It is also known that IVM binds to the transmembrane domains (TMs) of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. In this study, we found that the G-protein-gated inwardly rectifying K+ (GIRK) channel is activated by IVM directly. Electrophysiological recordings in Xenopus oocytes revealed that IVM activates GIRK channel in a phosphatidylinositol-4,5-biphosphate (PIP2 )-dependent manner, and that the IVM-mediated GIRK activation is independent of Gßγ subunits. We found that IVM activates GIRK2 more efficiently than GIRK4. In cultured hippocampal neurons, we also observed that IVM activates native GIRK current. Chimeric and mutagenesis analyses identified an amino acid residue unique to GIRK2 among the GIRK family, Ile82, located in the slide helix between the TM1 and the N-terminal cytoplasmic tail domain (CTD), which is critical for the activation. The results demonstrate that the TM-CTD interface in GIRK channels, rather than the TMs, governs IVM-mediated activation. These findings provide us with novel insights on the mode of action of IVM in ion channels that could lead to identification of new pharmacophores which activate the GIRK channel.

Chemical and thermal sensitivity of medaka TRPA1 analyzed in heterologous expression system.

TRPA1 of insects and several tetrapod vertebrates except for those of rodents have been reported to be activated by noxious chemicals and also by high temperature with a relatively clear threshold. We previously analyzed the characteristics of two TRPA1 paralogs of zebrafish (zTRPA1a, b) and demonstrated that zTRPA1a is specialized for chemical sensing while zTRPA1b responds to thermal stimulations, that zTRPA1b responds to both cold and heat stimuli, and that heat stimulation gradually activates zTRPA1b without a clear threshold. In the medaka genome, a single TRPA1 (olTRPA1) gene is present. To examine if functional properties of olTRPA1 are similar to TRPA1 of land animals or either of zTRPA1a or zTRPA1b, we isolated a TRPA1 cDNA from medaka and performed functional analyses. OlTRPA1 showed a sensitivity to four noxious chemicals (allyl isothiocyanate, caffeine, carvacrol, methyl anthranilate). We observed that cold stimulation does not activate olTRPA1, but heat stimulation gradually activates olTRPA1 with an unclear threshold. Results suggested that a single TRPA1 functions as a chemical and thermal sensor in medaka, and that a gradual heat-activation without clear threshold might be a common feature for TRPA1 of fish living in water.

Kv4.2 and accessory dipeptidyl peptidase-like protein 10 (DPP10) subunit preferentially form a 4:2 (Kv4.2:DPP10) channel complex.

Kv4 is a member of the voltage-gated K(+) channel family and forms a complex with various accessory subunits. Dipeptidyl aminopeptidase-like protein (DPP) is one of the auxiliary subunits for the Kv4 channel. Although DPP has been well characterized and is known to increase the current amplitude and accelerate the inactivation and recovery from inactivation of Kv4 current, it remains to be determined how many DPPs bind to one Kv4 channel. To examine whether the expression level of DPP changes the biophysical properties of Kv4, we expressed Kv4.2 and DPP10 in different ratios in Xenopus oocytes and analyzed the currents under two-electrode voltage clamp. The current amplitude and the speed of recovery from inactivation of Kv4.2 changed depending on the co-expression level of DPP10. This raised the possibility that the stoichiometry of the Kv4.2-DPP10 complex is variable and affects the biophysical properties of Kv4.2. We next determined the stoichiometry of DPP10 alone by subunit counting using single-molecule imaging. Approximately 70% of the DPP10 formed dimers in the plasma membrane, and the rest existed as monomers in the absence of Kv4.2. We next determined the stoichiometry of the Kv4.2-DPP10 complex; Kv4.2-mCherry and mEGFP-DPP10 were co-expressed in different ratios and the stoichiometries of Kv4.2-DPP10 complexes were evaluated by the subunit counting method. The stoichiometry of the Kv4.2-DPP10 complex was variable depending on the relative expression level of each subunit, with a preference for 4:2 stoichiometry. This preference may come from the bulky dimeric structure of the extracellular domain of DPP10.

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins.

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.

Sensitivities of Two Zebrafish TRPA1 Paralogs to Chemical and Thermal Stimuli Analyzed in Heterologous Expression Systems.

Transient receptor potential A1 (TRPA1) is the only member of the mouse, chick, and frog TRPA family, whereas 2 paralogs (zTRPA1a and zTRPA1b) are present in zebrafish. We herein investigated functional differences in the 2 zebrafish TRPA1s. HEK293T cells were used as heterologous expression systems, and the sensitivities of these cells to 4 chemical irritants (allyl isothiocyanate [AITC], caffeine, auto-oxidized epigallocatechin gallate [EGCG], and hydrogen peroxide [H2O2]) were compared with Ca(2+) imaging techniques. Sensitivities to the activators for AITC, oxidized EGCG, and H2O2 were higher in cells expressing zTRPA1a than in those expressing zTRPA1b, whereas caffeine appeared to activate both cells equally. We also characterized the thermal sensitivity of Xenopus oocytes expressing each TRPA1 electrophysiologically using a 2-electrode voltage clamp. Although endogenous currents induced by a cold stimulation were observed in control oocytes in some batches, oocytes expressing zTRPA1b showed significantly stronger cold- and heat-induced responses. However, significant thermal activation was not observed in oocytes expressing zTRPA1a. The results obtained using in vitro expression systems suggest that zTRPA1a is specialized for chemical sensing, whereas zTRPA1b responds to thermal stimuli. Furthermore, characterization of the chimeric molecule of TRPA1a and 1b revealed the importance of the N-terminal region in chemical and thermal sensing by zTRPA1s.

On-site energy supply at synapses through monocarboxylate transporters maintains excitatory synaptic transmission.

ATP production through oxidative phosphorylation in the mitochondria is the most efficient way to provide energy to various energy-consuming activities of the neurons. These processes require a large amount of ATP molecules to be maintained. Of these, synaptic transmission is most energy consuming. Here we report that lactate transported through monocarboxylate transporters (MCTs) at excitatory synapses constitutively supports synaptic transmission, even under conditions in which a sufficient supply of glucose and intracellular ATP are present. We analyzed the effects of MCT inhibition on neuronal activities using whole-cell recordings in brain slices of rats in the nucleus of the solitary tract. MCT inhibitors (α-cyano-4-hydroxycinnamic acid (4-CIN), phloretin, and d-lactate) significantly decreased the amplitude of EPSCs without reducing release probability. Although 4-CIN significantly reduced currents mediated by heterologously expressed AMPA-Rs in oocytes (a novel finding in this study), the IC50 of the inhibitory effect on EPSC in brain slices was ∼3.8 times smaller than that on AMPA-R currents in oocytes. Removal of intracellular ATP significantly potentiated the inhibition of EPSC with 4-CIN in a manner that was counteracted by intracellular lactate addition. In addition, extracellular lactate rescued aglycemic suppression of EPSC, in a manner that was prevented by 4-CIN. Inhibition of MCTs also reduced NMDA-R-mediated EPSCs and, to a lesser extent, the IPSC. The reduction in EPSC amplitude by γ-d-glutamylglycine was enhanced by 4-CIN, suggesting also a decreased quantal content. We conclude that "on-site" astrocyte-neuron lactate transport to presynaptic and postsynaptic elements is necessary for the integrity of excitatory synaptic transmission.

KCNQ1 channel modulation by KCNE proteins via the voltage-sensing domain.

The gating of the KCNQ1 potassium channel is drastically regulated by auxiliary subunit KCNE proteins. KCNE1, for example, slows the activation kinetics of KCNQ1 by two orders of magnitude. Like other voltage-gated ion channels, the opening of KCNQ1 is regulated by the voltage-sensing domain (VSD; S1-S4 segments). Although it has been known that KCNE proteins interact with KCNQ1 via the pore domain, some recent reports suggest that the VSD movement may be altered by KCNE. The altered VSD movement of KCNQ1 by KCNE proteins has been examined by site-directed mutagenesis, the scanning cysteine accessibility method (SCAM), voltage clamp fluorometry (VCF) and gating charge measurements. These accumulated data support the idea that KCNE proteins interact with the VSDs of KCNQ1 and modulate the gating of the KCNQ1 channel. In this review, we will summarize recent findings and current views of the KCNQ1 modulation by KCNE via the VSD. In this context, we discuss our recent findings that KCNE1 may alter physical interactions between the S4 segment (VSD) and the S5 segment (pore domain) of KCNQ1. Based on these findings from ourselves and others, we propose a hypothetical mechanism for how KCNE1 binding alters the VSD movement and the gating of the channel.

The stoichiometry and biophysical properties of the Kv4 potassium channel complex with K+ channel-interacting protein (KChIP) subunits are variable, depending on the relative expression level.

Kv4 is a voltage-gated K(+) channel, which underlies somatodendritic subthreshold A-type current (ISA) and cardiac transient outward K(+) (Ito) current. Various ion channel properties of Kv4 are known to be modulated by its auxiliary subunits, such as K(+) channel-interacting protein (KChIP) or dipeptidyl peptidase-like protein. KChIP is a cytoplasmic protein and increases the current amplitude, decelerates the inactivation, and accelerates the recovery from inactivation of Kv4. Crystal structure analysis demonstrated that Kv4 and KChIP form an octameric complex with four Kv4 subunits and four KChIP subunits. However, it remains unknown whether the Kv4·KChIP complex can have a different stoichiometry other than 4:4. In this study, we expressed Kv4.2 and KChIP4 with various ratios in Xenopus oocytes and observed that the biophysical properties of Kv4.2 gradually changed with the increase in co-expressed KChIP4. The tandem repeat constructs of Kv4.2 and KChIP4 revealed that the 4:4 (Kv4.2/KChIP4) channel shows faster recovery than the 4:2 channel, suggesting that the biophysical properties of Kv4.2 change, depending on the number of bound KChIP4s. Subunit counting by single-molecule imaging revealed that the bound number of KChIP4 in each Kv4.2·KChIP4 complex was dependent on the expression level of KChIP4. Taken together, we conclude that the stoichiometry of Kv4·KChIP complex is variable, and the biophysical properties of Kv4 change depending on the number of bound KChIP subunits.