Double gaps along Shaker S4 demonstrate omega currents at three different closed states

The aim of the present study was to investigate in detail how the voltage sensor in the Shaker potassium channel moves during the gating process. After the publication of the open channel structure from the crystallized KvAP channel in 2003, an alternative so-called "paddle" model was put forward in contrast to the existing helical screw model. The voltage sensor S4 contains 4 arginine residues relevant for gating, R1(362), R2(365), R3(368) and R4(371), each separated by 2 neutral residues. These charged residues coil as one of three threads on the S4-α-helix. Based on a previous finding that the mutation R1S leads to the so-called omega leak current through a "gating-pore" in the closed state, we introduced gaps systematically along the arginine thread substituting long arginines by short serines. Mutations R2S or R3S did neither create transient nor steady leaks. The fact that the native residue A359, which is located three amino acids in front of R1, is a short one, motivated us to check its role. Mutation of A359 to arginine blocked the omega current in R1S mutant indicating that the omega pore is occupied by A359 and R1. Introducing further double gaps (RR to SS) at sequential positions (0+1, 1+2, 2+3), produced clear leak currents which were remarkably stable over a wide voltage range. These leaks contradict that S4 would swing together with S3 in lipid according to the paddle hypothesis. Rather, our results show that during gating the S4 segment moves in 3 helical steps through a fixed pore formed by the channel protein.     

Functional extension of amino acid triads from the fourth transmembrane segment (S4) into its external linker in Shaker K(+) channels

The highly conserved fourth transmembrane segment (S4) is the primary voltage sensor of the voltage-dependent channel and would move outward upon membrane depolarization. S4 comprises repetitive amino acid triads, each containing one basic (presumably charged and voltage-sensing) followed by two hydrophobic residues. We showed that the triad organization is functionally extended into the S3-4 linker right external to S4 in Shaker K(+) channels. The arginine (and lysine) substitutes for the third and the sixth residues (Ala-359 and Met-356, respectively) external to the outmost basic residue (Arg-362) in S4 dramatically and additively stabilize S4 in the resting conformation. Also, Leu-361 and Leu-358 play a very similar role in stabilization of S4 in the resting position, presumably by their hydrophobic side chains. Moreover, the double mutation A359R/E283A leads to a partially extruded position of S4 and consequently prominent closed-state inactivation, suggesting that Glu-283 in S2 may coordinate with the arginines in the extruded S4 upon depolarization. We conclude that the triad organization extends into the S3-4 linker for about six amino acids in terms of their microenvironment. These approximately six residues should retain the same helical structure as S4, and their microenvironment serves as part of the "gating canal" accommodating the extruding S4. Upon depolarization, S4 most likely moves initially as a sliding helix and follows the path that is set by the approximately six residues in the S3-4 linker in the resting state, whereas further S4 translocation could be more like, for example, a paddle, without orderly coordination from the contiguous surroundings.     

Dynamics of the Kv1.2 voltage-gated K+ channel in a membrane environment

All-atom molecular dynamics simulations are used to better understand the dynamic environment experienced by the Kv1.2 channel in a lipid membrane. The structure of the channel is stable during the trajectories. The pore domain keeps a well-defined conformation, whereas the voltage-sensing domains undergo important lateral fluctuations, consistent with their modular nature. A channel-like region at the center of the S1-S4 helical bundle fills rapidly with water, reminiscent of the concept of high-dielectric aqueous crevices. The first two arginines along S4 (R294 and R297) adopt an interfacial position where they interact favorably with water and the lipid headgroups. The following two arginines (R300 and R303) interact predominantly with water and E226 in S2. Despite the absence of a structurally permanent gating pore formed by protein residues and surrounding the S4 helix, as traditionally pictured, the charged residues are located in a favorable environment and are not extensively exposed to the membrane nonpolar region. Continuum electrostatic computations indicate that the transmembrane potential sensed by the charged residues in the voltage sensor varies abruptly over the outer half of the membrane in the arginine-rich region of S4; thus, the voltage gradient or membrane electric field is "focused". Interactions of basic residues with the lipid headgroups at the intracellular membrane-solution interface reduce the membrane thickness near the channel, resulting in an increased transmembrane field.     

A direct demonstration of closed-state inactivation of K+ channels at low pH

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K⁺ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.     

Identification of positively charged residues of FomA porin of Fusobacterium nucleatum which are important for pore function.

FomA porin is the major outer-membrane protein of Fusobacterium nucleatum. It exhibits the functional properties of a general diffusion porin, but has no sequence similarity to other porins. According to the proposed topology model, each monomer of this trimeric protein is a beta-barrel consisting of 16 transmembrane segments with eight surface-exposed loops. Several conserved charged residues are proposed to extend from the beta-barrel wall into the aqueous channel lumen, and may contribute to a transverse electric field similar to that at the pore constriction of porins with known structure. The goal of our study was to identify particular basic residues contributing to such an electric field in FomA. Several arginines and lysines were replaced by negatively charged glutamates or uncharged alanines. The mutated FomA porins were expressed in Escherichia coli, and the effects on pore function were studied in vivo, by assaying the uptake rate of beta-lactam antibiotics, and in vitro after reconstitution of the purified proteins in lipid bilayer membranes. Some of the point mutations had a significant impact on the channel properties. The substitution R92A produced a 130% increased permeability of the zwitterionic beta-lactam cephaloridine, and the cation selectivity of R92E increased by 70%. The effects of the R90E substitution on channel properties were similar. Most of the point mutations had a minor effect on the voltage gating of the FomA channel, resulting in an increased sensitivity, except for K78E, which showed a decreased sensitivity. The latter mutation had no effect on cation selectivity, but the K78A substitution improved the uptake rate of cephaloridine. The results presented here indicate that arginines 90 and 92 are probably part of the constriction zone of the FomA porin, and lysine 78 and arginines 115 and 117 are probably in close proximity to this region as well.     

Role of hydrophobic residues in the voltage sensors of the voltage-gated sodium channel

The role of hydrophobic residues in voltage sensors S4 of voltage-sensitive ion channels is less documented than that of charged residues. We performed alanine-substitution of branched-sidechain residues contiguous to the third, fourth and fifth positively charged residues in S4s of the first three domains of the sodium channel expressed in HEK cells. These locations were selected because they are close to the arginines and lysines important in gating. Mutations in the first two domains (DIS4 and DIIS4) altered steady-state activation curves. In DIIIS4, the mutation L1131A next to the third arginine greatly slowed inactivation in a manner similar to that for substitutions of charged residues in DIVS4, whereas the mutation L1137A next to the fifth arginine preserved wild-type behaviour. Homology models of domain III, based on the structure of a crystallized mammalian potassium channel, shows that L1131 is located at the interface between S3 and S4 helices, whereas L1137, on the opposite side of S4, does not interact with the voltage sensor. The two mutated residues are closer to each other in domains I and II than in domain III, as may be corroborated by their different electrophysiological effects.     

Role of hydrophobic residues in the voltage sensors of the voltage-gated sodium channel

The role of hydrophobic residues in voltage sensors S4 of voltage-sensitive ion channels is less documented than that of charged residues. We performed alanine-substitution of branched-sidechain residues contiguous to the third, fourth and fifth positively charged residues in S4s of the first three domains of the sodium channel expressed in HEK cells. These locations were selected because they are close to the arginines and lysines important in gating. Mutations in the first two domains (DIS4 and DIIS4) altered steady-state activation curves. In DIIIS4, the mutation L1131A next to the third arginine greatly slowed inactivation in a manner similar to that for substitutions of charged residues in DIVS4, whereas the mutation L1137A next to the fifth arginine preserved wild-type behaviour. Homology models of domain III, based on the structure of a crystallized mammalian potassium channel, shows that L1131 is located at the interface between S3 and S4 helices, whereas L1137, on the opposite side of S4, does not interact with the voltage sensor. The two mutated residues are closer to each other in domains I and II than in domain III, as may be corroborated by their different electrophysiological effects.     

S4 charges move close to residues in the pore domain during activation in a K channel.

Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET(+)). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 A) to a position close (8 A) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.     

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.     

Coupling between Residues on S4 and S1 Defines the Voltage-Sensor Resting Conformation in NaChBac

The voltage sensor is a four-transmembrane helix bundle (S1-S4) that couples changes in membrane potential to conformational alterations in voltage-gated ion channels leading to pore opening and ion conductance. Although the structure of the voltage sensor in activated potassium channels is available, the conformation of the voltage sensor at rest is still obscure, limiting our understanding of the voltage-sensing mechanism. By employing a heterologously expressed Bacillus halodurans sodium channel (NaChBac), we defined constraints that affect the positioning and depolarization-induced outward motion of the S4 segment. We compared macroscopic currents mediated by NaChBac and mutants in which E43 on the S1 segment and the two outermost arginines (R1 and R2) on S4 were substituted. Neutralization of the negatively charged E43 (E43C) had a significant effect on channel gating. A double-mutant cycle analysis of E43 and R1 or R2 suggested changes in pairing during channel activation, implying that the interaction of E43 with R1 stabilizes the voltage sensor in its closed/available state, whereas interaction of E43 with R2 stabilizes the channel open/unavailable state. These constraints on S4 dynamics that define its stepwise movement upon channel activation and positioning at rest are novel, to the best of our knowledge, and compatible with the helical-screw and electrostatic models of S4 motion.     

The N-terminus of the K channel KAT1 controls its voltage-dependent gating by altering the membrane electric field.

Functional roles of different domains (pore region, S4 segment, N-terminus) of the KAT1 potassium channel in its voltage-dependent gating were electrophysiologically studied in Xenopus oocytes. The KAT1 properties did not depend on the extracellular K+ concentration or on residue H267, equivalent to one of the residues known to be important in C-type inactivation in Shaker channels, indicating that the hyperpolarization-induced KAT1 inward currents are related to the channel activation rather than to recovery from inactivation. Neutralization of a positively charged amino acid in the S4 domain (R176S) reduced the gating charge movement, suggesting that it acts as a voltage-sensing residue in KAT1. N-terminal deletions alone (e.g., delta20-34) did not affect the gating charge movement. However, the deletions paradoxically increased the voltage sensitivity of the R176S mutant channel, but not that of the wild-type channel. We propose a simple model in which the N-terminus determines the KAT1 voltage sensitivity by contributing to the electric field sensed by the voltage sensor.     

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.     

Two arginines in the cytoplasmic C-terminal domain are essential for voltage-dependent regulation of A-type K+ current in the Kv4 channel subfamily

Contributions of the C-terminal domain of Kv4.3 to the voltage-dependent gating of A-type K+ current (IA) were examined by (i) making mutations in this region, (ii) heterologous expression in HEK293 cells, and (iii) detailed voltage clamp analyses. Progressive deletions of the C terminus of rat Kv4.3M (to amino acid 429 from the N terminus) did not markedly change the inactivation time course of IA but shifted the voltage dependence of steady state inactivation in the negative direction to a maximum of -17 mV. Further deletions (to amino acid 420) shifted this parameter in the positive direction, suggesting a critical role for the domain 429-420 in the voltage-dependent regulation of IA. There are four positively charged amino acids in this domain: Lys423, Lys424, Arg426, and Arg429. The replacement of the two arginines with alanines (R2A) resulted in -23 and -13 mV shifts of inactivation and activation, respectively. Additional replacement of the two lysines with alanines did not result in further shifts. Single replacements of R426A or R429A induced -15 and -10 mV shifts of inactivation, respectively. R2A did not significantly change the inactivation rate but did markedly change the voltage dependence of recovery from inactivation. These two arginines are conserved in Kv4 subfamily, and alanine replacement of Arg429 and Arg432 in Kv4.2 gave essentially the same results. These effects of R2A were not modulated by co-expression of the K+ channel beta subunit, KChIPs. In conclusion, the two arginines in the cytosolic C-terminal domain of alpha-subunits of Kv4 subfamily strongly regulate the voltage dependence of channel activation, inactivation, and recovery.     

Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel

Voltage-dependent ion channels open and conduct ions in response to changes in cell-membrane voltage. The voltage sensitivity of these channels arises from the motion of charged arginine residues located on the S4 helices of the channel's voltage sensors. In KvAP, a prokaryotic voltage-dependent K+ channel, the S4 helix forms part of a helical hairpin structure, the voltage-sensor paddle. We have measured the membrane depth of residues throughout the KvAP channel using avidin accessibility to different-length tethered biotin reagents. From these measurements, we have calibrated the tether lengths and derived the thickness of the membrane that forms a barrier to avidin penetration, allowing us to determine the magnitude of displacement of the voltage-sensor paddles during channel gating. Here we show that the voltage-sensor paddles are highly mobile compared to other regions of the channel and transfer the gating-charge arginines 15-20 A through the membrane to open the pore.     

Role of Arginine Residues on the S4 Segment of the <Emphasis Type="Italic">Bacillus halodurans</Emphasis> Na<Superscript>+</Superscript> Channel in Voltage-sensing

The one-domain voltage-gated sodium channel of Bacillus halodurans (NaChBac) is composed of six transmembrane segments (S1–S6) comprising a pore-forming region flanked by segments S5 and S6 and a voltage-sensing element composed of segment S4. To investigate the role of the S4 segment in NaChBac channel activation, we used the cysteine mutagenesis approach where the positive charges of single and multiple arginine (R) residues of the S4 segment were replaced by the neutrally charged amino acid cysteine (C). To determine whether it was the arginine residue itself or its positive charge that was involved in channel activation, arginine to lysine (R to K) mutations were constructed. Wild-type (WT) and mutant NaChBac channels were expressed in tsA201 cells and Na⁺ currents were recorded using the whole-cell configuration of the patch-clamp technique. The current/voltage (I-V) and conductance/voltage (G-V) relationships steady-state inactivation (h _∞) and recovery from inactivation were evaluated to determine the effects of the S4 mutations on the biophysical properties of the NaChBac channel. R to C on the S4 segment resulted in a slowing of both activation and inactivation kinetics. Charge neutralization of arginine residues mostly resulted in a shift toward more positive potentials of G-V and h _∞ curves. The G-V curve shifts were associated with a decrease in slope, which may reflect a decrease in the gating charge involved in channel activation. Single neutralization of R114, R117, or R120 by C resulted in a very slow recovery from inactivation. Double neutralization of R111 and R129 confirmed the role of R111 in activation and suggested that R129 is most probably not part of the voltage sensor. Most of the R to K mutants retained WT-like current kinetics but exhibited an intermediate G-V curve, a steady-state inactivation shifted to more hyperpolarized potentials, and intermediate time constants of recovery from inactivation. This indicates that R, at several positions, plays an important role in channel activation. The data are consistent with the notion that the S4 is most probably the voltage sensor of the NaChBac channel and that both positive charges and the nature of the arginine residues are essential for channel activation.This revised version was published online in June 2005 with a corrected cover date.     

Focused electric field across the voltage sensor of potassium channels

Voltage-gated ion channels respond to changes in membrane potential by movement of their voltage sensors across the electric field between cytoplasmic and extracellular solutions. The principal voltage sensors in these proteins are positively charged S4 segments. The absolute magnitude of S4 movement discriminates two competing classes of gating models. In one class, the movement is <10 Angstrom due to the fact that the electric field is focused by aqueous crevices in the channel protein. In an alternative model, based in part on the crystal structure of a potassium channel, the side chains of S4 arginines move their charges across the bilayer's electric field, a distance of >25 Angstrom. Here, using tethered charges attached to an S4 segment, we provide evidence that the electric field falls across a distance of <4 Angstrom, supporting a model in which the relative movement between S4 and the electric field is very small.     

Gating Properties of a Sodium Channel with Three Arginines Substituted by Histidines in the Central Part of Voltage Sensor S4D4

In voltage-dependent sodium channels there is some functional specialization of the four different S4 voltage sensors with regard to the gating process. Whereas the voltage sensors of domains 1 to 3 control activation gating, the movement of the voltage sensor of domain 4 (S4D4) is known to be tightly coupled to sodium channel inactivation, and there is some experimental evidence that S4D4 also participates in activation gating. To further explore its putative multifunctional role in the gating process, we changed the central part of S4D4 in rat brain IIA (rBIIA) sodium channels by the simultaneous replacement of the third (R1632), fourth (R1635) and fifth (R1638) arginine by histidine (mutation R3/4/5H). As a result, the time course of current decay observed in R3/4/5H was about three times slower, if compared to wild type (WT). On the other hand, the recovery, as well as the voltage dependence of fast inactivation, remained largely unaffected by the mutation. This suggests that at physiological pH (7.5) the effective charge of the voltage sensor was not significantly changed by the amino-acid substitutions. The well-known impact of site-3 toxin (ATX-II) on the inactivation was drastically reduced in R3/4/5H, without changing the toxin affinity of the channel. The activation kinetics of WT and R3/4/5H studied at low temperature (8 degrees C) were indistinguishable, while the inactivation time course of R3/4/5H was then clearly more slowed than in WT. These data suggest that the replacement of arginines by histidines in the central part of S4D4 clearly affects the movement of S4D4 without changing the activation kinetics.     

Gating of voltage-dependent potassium channels

Activation of voltage-dependent ion channels is primarily controlled by the applied potential difference across the membrane. For potassium channels the Drosophila Shaker channel has served as an archetype of all other potassium channels in studies of activation mechanisms. In the Shaker potassium channel much of the voltage sensitivity is conferred by the S4 transmembrane helix, which contains seven positively charged residues. During gating, the movement of these charges produces gating currents. Mutagenic and fluorescence studies indicate that four of these residues are particularly important and contribute to the majority of gating charge, R362, R365, R368 and R371. The channel is thought to dwell in several closed states prior to opening. Ionic-charge pairing with negatively charged residues in the S2 and S3 helices is thought to be important in regulating these closed states and detailed kinetic models have attempted to define the kinetics and charge of the transitions between these states. Neutral residues throughout the S4 and S5 helices are thought to control late steps in channel opening and may have important roles in modulating the stability of the open state and late closed states. In response to depolarization, the S4 helix is thought to undergo a rotational translation and this movement is also important in studies of the movement of the pore helices, S5 and S6, during opening. This review will examine residues that are important during activation as well as kinetic models that have attempted to quantitatively define the activation pathway of voltage-dependent potassium channels.     

Role of hydrophobic and ionic forces in the movement of S4 of the Shaker potassium channel

Abstract

Voltage-gated ion (K⁺, Na⁺, Ca²⁺) channels contain a pore domain (PD) surrounded by four voltage sensing domains (VSD). Each VSD is made up of four transmembrane helices, S1–S4. S4 contains 6–7 positively charged residues (arginine/lysine) separated two hydrophobic residues, whereas S1–S3 contribute to two negatively charged clusters. These structures are conserved among all members of the voltage-gated ion channel family and play essential roles in voltage gating. The role of S4 charged residues in voltage gating is well established: During depolarization, they move out of the membrane electric field, exerting a mechanical force on channel gates, causing them to open. However, the role of the intervening hydrophobic residues in voltage sensing is unclear. Here we studied the role of these residues in the prototypical Shaker potassium channel. We have altered the physicochemical properties of both charged and hydrophobic positions of S4 and examined the effect of these modifications on the gating properties of the channel. For this, we have introduced cysteines at each of these positions, expressed the mutants in Xenopus oocytes, and examined the effect of in situ addition of charge, via Cd²⁺, on channel gating by two-electrode voltage clamp. Our results reveal a face of the S4 helix (comprising residues L358, L361, R365 and R368) where introduction of charge at hydrophobic positions destabilises the closed state and removal of charges from charged positions has an opposite effect. We propose that hydrophobic residues play a crucial role in limiting gating to a physiological voltage range.     

Role of S3 and S4 transmembrane domain charged amino acids in channel biogenesis and gating of KCa2.3 and KCa3.1

The role of positively charged arginines in the fourth transmembrane domain (S4) and a single negatively charged amino acid in the third transmembrane domain (S3) on channel biogenesis and gating of voltage-gated K(+) channels (Kv) has been well established. Both intermediate (KCa3.1) and small (KCa2.x) conductance, Ca(2+)-activated K(+) channels have two conserved arginines in S4 and a single conserved glutamic acid in S3, although these channels are voltage-independent. We demonstrate that mutation of any of these charged amino acids in KCa3.1 or KCa2.3 to alanine, glutamine, or charge reversal mutations results in a rapid degradation (<30 min) of total protein, confirming the critical role of these amino acids in channel biogenesis. Mutation of the S4 arginine closest to the cytosolic side of KCa3.1 to histidine resulted in expression at the cell surface. Excised patch clamp experiments revealed that this Arg/His mutation had a dramatically reduced open probability (P(o)), relative to wild type channels. Additionally, we demonstrate, using a combination of short hairpin RNA, dominant negative, and co-immunoprecipitation studies, that both KCa3.1 and KCa2.3 are translocated out of the endoplasmic reticulum associated with Derlin-1. These misfolded channels are poly-ubiquitylated, recognized by p97, and targeted for proteasomal degradation. Our results suggest that S3 and S4 charged amino acids play an evolutionarily conserved role in the biogenesis and gating of KCa channels. Furthermore, these improperly folded K(+) channels are translocated out of the endoplasmic reticulum in a Derlin-1- and p97-dependent fashion, poly-ubiquitylated, and targeted for proteasomal degradation.