KCNE1 constrains the voltage sensor of Kv7.1 K+ channels

Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K(+) channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs) and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K(+) channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions.     

KCNE1 remodels the voltage sensor of Kv7.1 to modulate channel function

The KCNE1 auxiliary subunit coassembles with the Kv7.1 channel and modulates its properties to generate the cardiac I_Ks current. Recent biophysical evidence suggests that KCNE1 interacts with the voltage-sensing domain (VSD) of Kv7.1. To investigate the mechanism of how KCNE1 affects the VSD to alter the voltage dependence of channel activation, we perturbed the VSD of Kv7.1 by mutagenesis and chemical modification in the absence and presence of KCNE1. Mutagenesis of S4 in Kv7.1 indicates that basic residues in the N-terminal half (S4-N) and C-terminal half (S4-C) of S4 are important for stabilizing the resting and activated states of the channel, respectively. KCNE1 disrupts electrostatic interactions involving S4-C, specifically with the lower conserved glutamate in S2 (Glu¹⁷⁰ or E2). Likewise, Trp scanning of S4 shows that mutations to a cluster of residues in S4-C eliminate current in the presence of KCNE1. In addition, KCNE1 affects S4-N by enhancing MTS accessibility to the top of the VSD. Consistent with the structure of Kv channels and previous studies on the KCNE1-Kv7.1 interaction, these results suggest that KCNE1 alters the interactions of S4 residues with the surrounding protein environment, possibly by changing the protein packing around S4, thereby affecting the voltage dependence of Kv7.1.     

Mutations within the S4-S5 linker alter voltage sensor constraints in hERG K+ channels

Human ether-a-go-go related gene (hERG) channel gating is associated with slow activation, yet the mechanistic basis for this is unclear. Here, we examine the effects of mutation of a unique glycine residue (G546) in the S4-S5 linker on voltage sensor movement and its coupling to pore gating. Substitution of G546 with residues possessing different physicochemical properties shifted activation gating by ∼−50 mV (with the exception of G546C). With the activation shift taken into account, the time constant of activation was also accelerated, suggesting a stabilization of the closed state by ∼1.6-4.3 kcal/mol (the energy equivalent of one to two hydrogen bonds). Predictions of the α-helical content of the S4-S5 linker suggest that the presence of G546 in wild-type hERG provides flexibility to the helix. Deactivation gating was affected differentially by the G546 substitutions. G546V induced a pronounced slow component of closing that was voltage-independent. Fluorescence measurements of voltage sensor movement in G546V revealed a slow component of voltage sensor return that was uncoupled from charge movement, suggesting a direct effect of the mutation on voltage sensor movement. These data suggest that G546 plays a critical role in channel gating and that hERG channel closing involves at least two independently modifiable reconfigurations of the voltage sensor.     

Mechanism of the modulation of Kv4:KChIP-1 channels by external K+

In response to a prolonged membrane depolarization, inactivation autoregulates the activity of voltage-gated ion channels. Slow inactivation involving a localized constriction of the selectivity filter (P/C-type mechanism) is prevalent in many voltage-gated K⁺ channels of the Kv1 subfamily. However, the generalization of this mechanism to other Kv channel subfamilies has remained uncertain and controversial. In agreement with a “foot-in-the-door” mechanism and the presence of ion-ion interactions in the pore, elevated external K⁺ slows the development of P/C-type inactivation and accelerates its recovery. In sharp contrast and resembling the regulation of the hippocampal A-type K⁺ current, we found that Kv4.x channels associated with KChIP-1 (an auxiliary subunit) exhibit accelerated inactivation and unaffected recovery from inactivation when exposed to elevated external K⁺. This regulation depends on the ability of a permeant ion to enter the selectivity filter (K⁺ = Rb⁺ = NH4⁺ > Cs⁺ > Na⁺); and the apparent equilibrium dissociation constant of a single regulatory site is 8 mM for K⁺. By applying a robust quantitative global kinetic modeling approach to all macroscopic properties over a 210-mV range of membrane potentials, we determined that elevated external K⁺ inhibits unstable closed states outside the main activation pathway and thereby promotes preferential closed-state inactivation. These results suggest the presence of a vestigial and unstable P/C-type mechanism of inactivation in Kv4 channels and strengthen the concept of novel mechanisms of closed-state inactivation. Regulation of Kv4 channel inactivation by hyperkalemia may help to explain the pathophysiology of electrolyte imbalances in excitable tissues.     

Role of charged residues in the S1-S4 voltage sensor of BK channels

The activation of large conductance Ca(2+)-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K(+) (K(V)) channels. Yet BK and K(V) channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (P(o)) and I(K) kinetics (tau(I(K))) over an extended voltage range in 0-50 microM [Ca(2+)](i). mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of P(O). The voltage dependence of P(O) and tau(I(K)) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and K(V) channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to K(V) channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol(-1), indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.     

Extent of voltage sensor movement during gating of shaker K+ channels

Voltage-driven activation of Kv channels results from conformational changes of four voltage sensor domains (VSDs) that surround the K(+) selective pore domain. How the VSD helices rearrange during gating is an area of active research. Luminescence resonance energy transfer (LRET) is a powerful spectroscopic ruler uniquely suitable for addressing the conformational trajectory of these helices. Using a geometric analysis of numerous LRET measurements, we were able to estimate LRET probe positions relative to existing structural models. The experimental movement of helix S4 does not support a large 15-20 A transmembrane "paddle-type" movement or a near-zero A vertical "transporter-type" model. Rather, our measurements demonstrate a moderate S4 displacement of 10 +/- 5 A, with a vertical component of 5 +/- 2 A. The S3 segment moves 2 +/- 1 A in the opposite direction and is therefore not moving as an S3-S4 rigid body.     

KCNE peptides differently affect voltage sensor equilibrium and equilibration rates in KCNQ1 K+ channels

KCNQ1 voltage-gated K(+) channels assemble with the family of KCNE type I transmembrane peptides to afford membrane-embedded complexes with diverse channel gating properties. KCNQ1/KCNE1 complexes generate the very slowly activating cardiac I(Ks) current, whereas assembly with KCNE3 produces a constitutively conducting complex involved in K(+) recycling in epithelia. To determine whether these two KCNE peptides influence voltage sensing in KCNQ1 channels, we monitored the position of the S4 voltage sensor in KCNQ1/KCNE complexes using cysteine accessibility experiments. A panel of KCNQ1 S4 cysteine mutants was expressed in Xenopus oocytes, treated with the membrane-impermeant cysteine-specific reagent 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and the voltage-dependent accessibility of each mutant was determined. Of these S4 cysteine mutants, three (R228C, G229C, I230C) were modified by MTSET only when KCNQ1 was depolarized. We then employed these state-dependent residues to determine how assembly with KCNE1 and KCNE3 affects KCNQ1 voltage sensor equilibrium and equilibration rates. In the presence of KCNE1, MTSET modification rates for the majority of the cysteine mutants were approximately 10-fold slower, as was recently reported to indicate that the kinetics of the KCNQ1 voltage sensor are slowed by KCNE1 (Nakajo, K., and Y. Kubo. 2007 J. Gen. Physiol. 130:269-281). Since MTS modification rates reflect an amalgam of reagent accessibility, chemical reactivity, and protein conformational changes, we varied the depolarization pulse duration to determine whether KCNE1 slows the equilibration rate of the voltage sensors. Using the state-dependent cysteine mutants, we determined that MTSET modification rates were essentially independent of depolarization pulse duration. These results demonstrate that upon depolarization the voltage sensors reach equilibrium quickly in the presence of KCNE1 and the slow gating of the channel complex is not due to slowly moving voltage sensors. In contrast, all cysteine substitutions in the S4 of KCNQ1/KCNE3 complexes were freely accessible to MTSET independent of voltage, which is consistent with KCNE3 shifting the voltage sensor equilibrium to favor the active state at hyperpolarizing potentials. In total, these results suggest that KCNE peptides differently modulate the voltage sensor in KCNQ1 K(+) channels.     

N-terminal PDZ-binding domain in Kv1 potassium channels

We have investigated the interactions of prototypical PDZ domains with both the C- and N-termini of Kv1.5 and other Kv channels. A combination of in vitro binding and yeast two-hybrid assays unexpectedly showed that PDZ domains derived from PSD95 bind both the C- and N-termini of the channels with comparable avidity. From doubly transfected HEK293 cells, Kv1.5 was found to co-immunoprecipitate with the PDZ protein, irrespective of the presence of the canonical C-terminal PDZ-binding motif in Kv1.5. Imaging analysis of the same HEK cell lines demonstrated that co-localization of Kv1.5 with PSD95 at the cell surface is similarly independent of the canonical PDZ-binding motif. Deletion analysis localized the N-terminal PDZ-binding site in Kv1.5 to the T1 region of the channel. Co-expression of PSD95 with Kv1.5 N- and C-terminal deletions in HEK cells had contrasting effects on the magnitudes of the potassium currents across the membranes of these cells. These findings may have important implications for the regulation of channel expression and function by PDZ proteins like PSD95.     

State-dependent electrostatic interactions of S4 arginines with E1 in S2 during Kv7.1 activation

The voltage-sensing domain of voltage-gated channels is comprised of four transmembrane helices (S1–S4), with conserved positively charged residues in S4 moving across the membrane in response to changes in transmembrane voltage. Although it has been shown that positive charges in S4 interact with negative countercharges in S2 and S3 to facilitate protein maturation, how these electrostatic interactions participate in channel gating remains unclear. We studied a mutation in Kv7.1 (also known as KCNQ1 or KvLQT1) channels associated with long QT syndrome (E1K in S2) and found that reversal of the charge at E1 eliminates macroscopic current without inhibiting protein trafficking to the membrane. Pairing E1R with individual charge reversal mutations of arginines in S4 (R1–R4) can restore current, demonstrating that R1–R4 interact with E1. After mutating E1 to cysteine, we probed E1C with charged methanethiosulfonate (MTS) reagents. MTS reagents could not modify E1C in the absence of KCNE1. With KCNE1, (2-sulfonatoethyl) MTS (MTSES)⁻ could modify E1C, but [2-(trimethylammonium)ethyl] MTS (MTSET)⁺ could not, confirming the presence of a positively charged environment around E1C that allows approach by MTSES⁻ but repels MTSET⁺. We could change the local electrostatic environment of E1C by making charge reversal and/or neutralization mutations of R1 and R4, such that MTSET⁺ modified these constructs depending on activation states of the voltage sensor. Our results confirm the interaction between E1 and the fourth arginine in S4 (R4) predicted from open-state crystal structures of Kv channels and reveal an E1–R1 interaction in the resting state. Thus, E1 engages in electrostatic interactions with arginines in S4 sequentially during the gating movement of S4. These electrostatic interactions contribute energetically to voltage-dependent gating and are important in setting the limits for S4 movement.     

Subunit-specific effect of the voltage sensor domain on Ca2+ sensitivity of BK channels

Large conductance Ca²⁺- and voltage-activated K⁺ (BK) channels, composed of pore-forming α-subunits and auxiliary β-subunits, play important roles in diverse physiological processes. The differences in BK channel phenotypes are primarily due to the tissue-specific expression of β-subunits (β1-β4) that modulate channel function differently. Yet, the molecular basis of the subunit-specific regulation is not clear. In our study, we demonstrate that perturbation of the voltage sensor in BK channels by mutations selectively disrupts the ability of the β1-subunit—but not that of the β2-subunit—to enhance apparent Ca²⁺ sensitivity. These mutations change the number of equivalent gating charges, the voltage dependence of voltage sensor movements, the open-close equilibrium of the channel, and the allosteric coupling between voltage sensor movements and channel opening to various degrees, indicating that they alter the conformation and movements of the voltage sensor and the activation gate. Similarly, the ability of the β1-subunit to enhance apparent Ca²⁺ sensitivity is diminished to various degrees, correlating quantitatively with the shift of voltage dependence of voltage sensor movements. In contrast, none of these mutations significantly reduces the ability of the β2-subunit to enhance Ca²⁺ sensitivity. These results suggest that the β1-subunit enhances Ca²⁺ sensitivity by altering the conformation and movements of the voltage sensor, whereas the similar function of the β2-subunit is governed by a distinct mechanism.     

The S4 voltage sensor packs against the pore domain in the KAT1 voltage-gated potassium channel

In voltage-gated ion channels, the S4 transmembrane segment responds to changes in membrane potential and controls channel opening. The local environment of S4 is still unknown, even regarding the basic question as to whether S4 is close to the pore domain. Relying on the ability of functional KAT1 channels to rescue potassium (K+) transport-deficient yeast, we have performed an unbiased mutagenesis screen aimed at determining whether S4 packs against S5 of the pore domain. Starting with semilethal mutations of surface-exposed S5 residues of the KAT1 pore domain, we have screened randomly mutagenized libraries of S4 or S1-S3 for second-site suppressors. Our study identifies two S4 residues that interact in a highly specific manner with two S5 residues in the middle of the membrane-spanning regions, supporting a model in which the S4 voltage sensor packs against the pore domain in the hyperpolarized, or "down," state of S4.     

External pH modulates EAG superfamily K+ channels through EAG-specific acidic residues in the voltage sensor

The Ether-a-go-go (EAG) superfamily of voltage-gated K⁺ channels consists of three functionally distinct gene families (Eag, Elk, and Erg) encoding a diverse set of low-threshold K⁺ currents that regulate excitability in neurons and muscle. Previous studies indicate that external acidification inhibits activation of three EAG superfamily K⁺ channels, Kv10.1 (Eag1), Kv11.1 (Erg1), and Kv12.1 (Elk1). We show here that Kv10.2, Kv12.2, and Kv12.3 are similarly inhibited by external protons, suggesting that high sensitivity to physiological pH changes is a general property of EAG superfamily channels. External acidification depolarizes the conductance–voltage (GV) curves of these channels, reducing low threshold activation. We explored the mechanism of this high pH sensitivity in Kv12.1, Kv10.2, and Kv11.1. We first examined the role of acidic voltage sensor residues that mediate divalent cation block of voltage activation in EAG superfamily channels because protons reduce the sensitivity of Kv12.1 to Zn²⁺. Low pH similarly reduces Mg²⁺ sensitivity of Kv10.1, and we found that the pH sensitivity of Kv11.1 was greatly attenuated at 1 mM Ca²⁺. Individual neutralizations of a pair of EAG-specific acidic residues that have previously been implicated in divalent block of diverse EAG superfamily channels greatly reduced the pH response in Kv12.1, Kv10.2, and Kv11.1. Our results therefore suggest a common mechanism for pH-sensitive voltage activation in EAG superfamily channels. The EAG-specific acidic residues may form the proton-binding site or alternatively are required to hold the voltage sensor in a pH-sensitive conformation. The high pH sensitivity of EAG superfamily channels suggests that they could contribute to pH-sensitive K⁺ currents observed in vivo.     

Nesfatin-1 inhibits voltage gated K+ channels in pancreatic beta cells

The anorexigenic neuropeptide NEFA/nucleobindin 2 (NUCB2)/nesfatin-1-containing neurons are distributed in the brain regions involved in feeding regulation. In spite of the growing knowledge of its physiological functions through extensive studies, its molecular mechanism of reaction, including its receptor, remains unknown. NUCB2/nesfatin-1 is also involved in various peripheral regulations, including glucose homeostasis. In pancreatic beta-cells, NUCB2/nesfatin-1 is reported to enhance glucose-stimulated insulin secretion (GSIS) but its exact mechanism remains unknown.To clarify this mechanism, we measured the effect of nesfatin-1 on the electrical activity of pancreatic beta-cells. Using mouse primary beta cells, we measured changes in the ATP-sensitive K⁺ (K_ATP) channel current, the voltage-gated K⁺ (Kv) channel current, and insulin secretion upon application of nesfatin-1.Nesfatin-1 inhibited the Kv channel, but K_ATP channel activity was unaffected. Nesfatin-1 enhanced insulin secretion to a same level as Kv channel blocker tetraethylammonium (TEA). The effect was not further enhanced when nesfatin-1 and TEA were applied simultaneously. The inhibition binding assay with [¹²⁵I]nesfatin-1 in Kv2.1 channels, major contributor of Kv current in beta cell, expressing HEK239 cells indicated the binding of nesfatin-1 on Kv2.1 channel.Because Kv channel inhibition enhances insulin secretion under high glucose conditions, our present data suggest a possible mechanism of nesfatin-1 on enhancing GSIS through regulation of ion channels rather than its unidentified receptor.     

Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway

The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.     

Interactions between S4-S5 linker and S6 transmembrane domain modulate gating of HERG K+ channels

Outward movement of the voltage sensor is coupled to activation in voltage-gated ion channels; however, the precise mechanism and structural basis of this gating event are poorly understood. Potential insight into the coupling mechanism was provided by our previous finding that mutation to Lys of a single residue (Asp(540)) located in the S4-S5 linker endowed HERG (human ether-a-go-go-related gene) K(+) channels with the unusual ability to open in response to membrane depolarization and hyperpolarization in a voltage-dependent manner. We hypothesized that the unusual hyperpolarization-induced gating occurred through an interaction between Lys(540) and the C-terminal end of the S6 domain, the region proposed to form the activation gate. Therefore, we mutated six residues located in this region of S6 (Ile(662)-Tyr(667)) to Ala in D540K HERG channels. Mutation of Arg(665), but not the other five residues, prevented hyperpolarization-dependent reopening of D540K HERG channels. Mutation of Arg(665) to Gln or Asp also prevented reopening. In addition, D540R and D540K/R665K HERG reopened in response to hyperpolarization. Together these findings suggest that a single residue (Arg(665)) in the S6 domain interacts with Lys(540) by electrostatic repulsion to couple voltage sensing to hyperpolarization-dependent opening of D540K HERG K(+) channels. Moreover, our findings suggest that the C-terminal ends of S4 and S6 are in close proximity at hyperpolarized membrane potentials.     

Structural compatibility between the putative voltage sensor of voltage-gated K+ channels and the prokaryotic KcsA channel

Sequence similarity among and electrophysiological studies of known potassium channels, along with the three-dimensional structure of the Streptomyces lividans K(+) channel (KcsA), support the tenet that voltage-gated K(+) channels (Kv channels) consist of two distinct modules: the "voltage sensor" module comprising the N-terminal portion of the channel up to and including the S4 transmembrane segment and the "pore" module encompassing the C-terminal portion from the S5 transmembrane segment onward. To substantiate this modular design, we investigated whether the pore module of Kv channels may be replaced with the pore module of the prokaryotic KcsA channel. Biochemical and immunocytochemical studies showed that chimeric channels were expressed on the cell surface of Xenopus oocytes, demonstrating that they were properly synthesized, glycosylated, folded, assembled, and delivered to the plasma membrane. Unexpectedly, surface-expressed homomeric chimeras did not exhibit detectable voltage-dependent channel activity upon both hyperpolarization and depolarization regardless of the expression system used. Chimeras were, however, strongly dominant-negative when coexpressed with wild-type Kv channels, as evidenced by the complete suppression of wild-type channel activity. Notably, the dominant-negative phenotype correlated well with the formation of stable, glycosylated, nonfunctional, heteromeric channels. Collectively, these findings imply a structural compatibility between the prokaryotic pore module and the eukaryotic voltage sensor domain that leads to the biogenesis of non-responsive channels. Our results lend support to the notion that voltage-dependent channel gating depends on the precise coupling between both protein domains, probably through a localized interaction surface.     

Autonomous transmembrane segment S4 of the voltage sensor domain partitions into the lipid membrane

The S4 transmembrane segment in voltage-gated ion channels, a highly basic α helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2±0.2kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9?. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the α helicity and regular spacing of basic amino-acid residues in the S4 sequence.     

Arrangement and mobility of the voltage sensor domain in prokaryotic voltage-gated sodium channels

Prokaryotic voltage-gated sodium channels (Na(V)s) form homotetramers with each subunit contributing six transmembrane α-helices (S1-S6). Helices S5 and S6 form the ion-conducting pore, and helices S1-S4 function as the voltage sensor with helix S4 thought to be the essential element for voltage-dependent activation. Although the crystal structures have provided insight into voltage-gated K channels (K(V)s), revealing a characteristic domain arrangement in which the voltage sensor domain of one subunit is close to the pore domain of an adjacent subunit in the tetramer, the structural and functional information on Na(V)s remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic Na(V), is similar to that in K(V)s. Cysteine substitutions of three residues in helix S4, Q107C, T110C, and R113C, effectively induced intersubunit disulfide bond formation with a cysteine introduced in helix S5, M164C, of the adjacent subunit. In addition, substituting two acidic residues with lysine, E43K and D60K, shifted the activation of the channel to more positive membrane potentials and consistently shifted the preferentially formed disulfide bond from T110C/M164C to Q107C/M164C. Because Gln-107 is located closer to the extracellular side of helix S4 than Thr-110, this finding suggests that the functional shift in the voltage dependence of activation is related to a restriction of the position of helix S4 in the lipid bilayer. The domain arrangement and vertical mobility of helix S4 in NaChBac indicate that the structure and the mechanism of voltage-dependent activation in prokaryotic Na(V)s are similar to those in canonical K(V)s.     

Differential association of the auxiliary subunit Kvbeta2 with Kv1.4 and Kv4.3 K+ channels

Kvbeta2 subunits associate with Kv1 and Kv4 K+ channels, but the basis of preferential association is not understood. For example, detergent resistance suggests stronger auxiliary subunit association with Kv4.2 than with Kv1.2, but Kvbeta2 preferentially localizes with the latter channels in brain. Here we examine the interaction of Kvbeta2 with two native binding partners in brain: Kv4.3 and Kv1.4. We show that the auxiliary subunit binds more efficiently to Kv1.4 than to Kv4.3 in mammalian cells. However, preexisting Kvbeta2 complexes with Kv1.4 and Kv4.3 have similar detergent sensitivity. Thus, preferential steady state binding may reflect a difference in initial association rather than stability. We also find that that the cytoplasmic C-terminus of Kv4.3 inhibits Kvbeta2 association. Apparently, a region proximal to the Kv4.3 pore contributes to the inefficient auxiliary subunit interaction that produces preferential binding of Kvbeta2 to Kv1 channels.     

KCNQ1 channels voltage dependence through a voltage-dependent binding of the S4-S5 linker to the pore domain

Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1–S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5–S6) is surrounded by four voltage sensor domains (S1–S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5_L) and S6 C terminus (S6_T). From these data, we hypothesized that S4S5_L behaves like a ligand specifically interacting with S6_T and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5_L and S6_T of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5_L peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5_L peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6_T, consistent with S4S5_L binding to S6_T. Val²⁵⁴ in S4S5_L is known to contact Leu³⁵³ in S6_T when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5_L binding to S6_T. Our results suggest a mechanistic model in which S4S5_L acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5_L away from S6_T, allowing channel opening.