Mutations in the KCNA1 gene associated with episodic ataxia type-1 syndrome impair heteromeric voltage-gated K(+) channel function.

Episodic ataxia type-1 syndrome (EA-1) is an autosomal dominant neurological disorder that manifests itself during infancy and results from point mutations in the voltage-gated potassium channel gene hKv1.1. The hallmark of the disease is continuous myokymia and episodic attacks of spastic contractions of the skeletal muscles, which cause permanent disability. Coexpression of hKv1.1 and hKv1.2 subunits produces heteromeric potassium channels with biophysical and pharmacological properties intermediate between the respective homomers. By using tandemly linked subunits, we demonstrate that hKv1.1 subunits bearing the EA-1 mutations V408A and E325D combine with hKv1.2 to produce channels with altered kinetics of activation, deactivation, C-type inactivation, and voltage dependence. Moreover, hKv1.1V408A single-channel analysis reveals a approximately threefold reduction of the mean open duration of the channel compared with the wild-type, and this mutation alters the open-state stability of both homomeric and heteromeric channels. The results demonstrate that human Kv1.2 and Kv1.1 subunits coassemble to form a novel channel with distinct gating properties that are altered profoundly by EA-1 mutations, thus uncovering novel physiopathogenetic mechanisms of episodic ataxia type-1 myokymia syndrome.     

Episodic ataxia type 1 mutations affect fast inactivation of K+ channels by a reduction in either subunit surface expression or affinity for inactivation domain

Episodic ataxia type 1 (EA1) is an autosomal dominant disorder characterized by continuous myokymia and episodic attacks of ataxia. Mutations in the gene KCNA1 that encodes the voltage-gated potassium channel Kv1.1 are responsible for EA1. In several brain areas, Kv1.1 coassembles with Kv1.4, which confers N-type inactivating properties to heteromeric channels. It is therefore likely that the rate of inactivation will be determined by the number of Kv1.4 inactivation particles, as set by the precise subunit stoichiometry. We propose that EA1 mutations affect the rate of N-type inactivation either by reduced subunit surface expression, giving rise to a reduced number of Kv1.1 subunits in heterotetramer Kv1.1-Kv1.4 channels, or by reduced affinity for the Kv1.4 inactivation domain. To test this hypothesis, quantified amounts of mRNA for Kv1.4 or Kv1.1 containing selected EA1 mutations either in the inner vestibule of Kv1.1 on S6 or in the transmembrane regions were injected into Xenopus laevis oocytes and the relative rates of inactivation and stoichiometry were determined. The S6 mutations, V404I and V408A, which had normal surface expression, reduced the rate of inactivation by a decreased affinity for the inactivation domain while the mutations I177N in S1 and E325D in S5, which had reduced subunit surface expression, increased the rate of N-type inactivation due to a stoichiometric increase in the number of Kv1.4 subunits.     

The Selectivity Filter Is Involved in the U-Type Inactivation Process of Kv2.1 and Kv3.1 Channels

Voltage-gated potassium (Kv) channels display several types of inactivation processes, including N-, C-, and U-types. C-type inactivation is attributed to a nonconductive conformation of the selectivity filter (SF). It has been proposed that the activation gate and the channel’s SF are allosterically coupled because the conformational changes of the former affect the structure of the latter and vice versa. The second threonine of the SF signature sequence (e.g., TTVGYG) has been proven to be essential for this allosteric coupling. To further study the role of the SF in U-type inactivation, we substituted the second threonine of the TTVGYG sequence by an alanine in the hKv2.1 and hKv3.1 channels, which are known to display U-type inactivation. Both hKv2.1-T377A and hKv3.1-T400A yielded channels that were resistant to inactivation, and as a result, they displayed noninactivating currents upon channel opening; i.e., hKv2.1-T377A and hKv3.1-T400A remained fully conductive upon prolonged moderate depolarizations, whereas in wild-type hKv2.1 and hKv3.1, the current amplitude typically reduces because of U-type inactivation. Interestingly, increasing the extracellular K⁺ concentration increased the macroscopic current amplitude of both hKv2.1-T377A and hKv3.1-T400A, which is similar to the response of the homologous T to A mutation in Shaker and hKv1.5 channels that display C-type inactivation. Our data support an important role for the second threonine of the SF signature sequence in the U-type inactivation gating of hKv2.1 and hKv3.1.     

Inactivation gating of Kv4 potassium channels: molecular interactions involving the inner vestibule of the pore

Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174- 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163-174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603-626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316-2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance-voltage curve ( approximately 5 mV) and the prepulse inactivation curve (>10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specific residue in the S4-S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404, 406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore's internal vestibule control both closing and inactivation in Kv4 K+ channels.     

Fast and slow voltage sensor movements in HERG potassium channels

HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH₂-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals.     

Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F

C-type inactivation in Shaker potassium channels inhibits K⁺ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na⁺ and Li⁺ currents in K⁺-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na⁺ and Li⁺ currents (in K⁺-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na⁺ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.     

Cooperative subunit interactions in C-type inactivation of K channels.

C-type inactivation of potassium channels is distinct from N-terminal mediated (N-type) inactivation and involves a closing of the outer mouth of the channel. We have investigated the role of the individual subunits of the tetrameric channel in the C-type inactivation conformational change by comparing the inactivation rates of channels constructed from different combinations of subunits. The relationship between the inactivation rate and the number of fast subunits is exponential, as would be predicted by a cooperative mechanism where the C-type conformational change involves all four subunits, and rules out a mechanism where a conformational change in any of the individual subunits is sufficient for inactivation. Subunit interactions in C-type inactivation are further supported by an interaction between separate mutations affecting C-type inactivation when in either the same or separate subunits.     

Non-Native R1 Substitution in the S4 Domain Uniquely Alters Kv4.3 Channel Gating

The S4 transmembrane domain in Shaker (Kv1) voltage-sensitive potassium channels has four basic residues (R1–R4) that are responsible for carrying the majority of gating charge. In Kv4 channels, however, R1 is replaced by a neutral valine at position 287. Among other differences, Kv4 channels display prominent closed state inactivation, a mechanism which is minimal in Shaker. To determine if the absence of R1 is responsible for important variation in gating characteristics between the two channel types, we introduced the V287R mutant into Kv4.3 and analyzed its effects on several voltage sensitive gating transitions. We found that the mutant increased the voltage sensitivity of steady-state activation and altered the kinetics of activation and deactivation processes. Although the kinetics of macroscopic inactivation were minimally affected, the characteristics of closed-state inactivation and recovery from open and closed inactivated states were significantly altered. The absence of R1 can only partially account for differences in the effective voltage sensitivity of gating between Shaker and Kv4.3. These results suggest that the S4 domain serves an important functional role in Kv4 channel activation and deactivation processes, and also those of closed-state inactivation and recovery.     

Long QT syndrome-associated mutations in the S4-S5 linker of KvLQT1 potassium channels modify gating and interaction with minK subunits.

Long QT syndrome is an inherited disorder of cardiac repolarization caused by mutations in cardiac ion channel genes, including KVLQT1. In this study, the functional consequences of three long QT-associated missense mutations in KvLQT1 (R243C, W248R, E261K) were characterized using the Xenopus oocyte heterologous expression system and two-microelectrode voltage clamp techniques. These mutations are located in or near the intracellular linker between the S4 and S5 transmembrane domains, a region implicated in activation gating of potassium channels. The E261K mutation caused loss of function and did not interact with wild-type KvLQT1 subunits. R243C or W248R KvLQT1 subunits formed functional channels, but compared with wild-type KvLQT1 current, the rate of activation was slower, and the voltage dependence of activation and inactivation was shifted to more positive potentials. Co expression of minK and KvLQT1 channel subunits induces a slow delayed rectifier K(+) current, I(Ks), characterized by slow activation and a markedly increased magnitude compared with current induced by KvLQT1 subunits alone. Coexpression of minK with R243C or W248R KvLQT1 subunits suppressed current, suggesting that coassembly of mutant subunits with minK prevented normal channel gating. The decrease in I(Ks) caused by loss of function or altered gating properties explains the prolonged QT interval and increased risk of arrhythmia and sudden death associated with these mutations in KVLQT1.     

Residues at the outer mouth of Kir1.1 determine K-dependent gating

Three residues (E132, F127, and R128) at the outer mouth of Kir1.1b directly affected inward rectifier gating by external K, independent of pH gating. Each of the individual mutations E132Q, F127V, F127D, and R128Y changed the normal K dependence of macroscopic conductance from hyperbolic (Km = 6 ± 2 mM) to linear, up to 500 mM, without changing the hyperbolic K dependence of single-channel conductance. This suggests that E132, F127, and R128 are responsible for maximal Kir1.1b activation by external K. In addition, these same residues were also essential for recovery of Kir1.1b activity after complete removal of external K by 18-Crown-6 polyether. In contrast, charge-altering mutations at neighboring residues (E92A, E104A, D97V, or Q133E) near the outer mouth of the channel did not affect Kir1.1b recovery after chelation of external K. The collective role of E132, R128, and F127 in preventing Kir1.1b inactivation by either cytoplasmic acidification or external K removal implies that pH inactivation and the external K sensor share a common mechanism, whereby E132, R128, and F127 stabilize the Kir1.1b selectivity filter gate in an open conformation, allowing rapid recovery of channel activity after a period of external K depletion.     

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Inactivation is an intrinsic property of numerous voltage-gated K⁺ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.     

Fast inactivation in potassium channels: An interplay of cytoplasmic domains

Fast inactivation in voltage-gated potassium channels has traditionally been associated exclusively with the N-terminus. Here, we explore the role of the T1 domain using a series of chimeric channels. A chimeric channel, 4N/2, (N-terminus from the rapidly inactivating hKv1.4, and the channel body from the non-inactivating hKv1.2), exhibited slower and incomplete inactivation as compared to the wild-type hKv1.4. Replacing the T1 domain of 4N2 with that from hKv1.2 (4N/2T1/2), restored inactivation, while that from hKv1.1 (4N/1T1/2) completely abolished inactivation. Based on these observations, we hypothesize a correlation between the tetramerization domain and the putative inactivation domain receptor in the process of rapid inactivation of hKv1 channels.     

Rescue of aberrant gating by a genetically encoded PAS (Per-Arnt-Sim) domain in several long QT syndrome mutant human ether-á-go-go-related gene potassium channels

Congenital long QT syndrome 2 (LQT2) is caused by loss-of-function mutations in the human ether-á-go-go-related gene (hERG) voltage-gated potassium (K(+)) channel. hERG channels have slow deactivation kinetics that are regulated by an N-terminal Per-Arnt-Sim (PAS) domain. Only a small percentage of hERG channels containing PAS domain LQT2 mutations (hERG PAS-LQT2) have been characterized in mammalian cells, so the functional effect of these mutations is unclear. We investigated 11 hERG PAS-LQT2 channels in HEK293 cells and report a diversity of functional defects. Most hERG PAS-LQT2 channels formed functional channels at the plasma membrane, as measured by whole cell patch clamp recordings and cell surface biotinylation. Mutations located on one face of the PAS domain (K28E, F29L, N33T, R56Q, and M124R) caused defective channel gating, including faster deactivation kinetics and less steady-state inactivation. Conversely, the other mutations caused no measurable differences in channel gating (G53R, H70R, and A78P) or no measurable currents (Y43C, C66G, and L86R). We used a genetically encoded hERG PAS domain (NPAS) to examine whether channel dysfunction could be corrected. We found that NPAS fully restored wild-type-like deactivation kinetics and steady-state inactivation to the hERG PAS-LQT2 channels. Additionally, NPAS rescued aberrant currents in hERG R56Q channels during a dynamic ramp voltage clamp. Thus, our results reveal a putative "gating face" in the PAS domain where mutations within this region form functional channels with altered gating properties, and we show that NPAS is a general means for rescuing aberrant gating in hERG LQT2 mutant channels and may be a potential biological therapeutic.     

Charge immobilization of skeletal muscle Na+ channels: role of residues in the inactivation linker

We investigated structural determinants of fast inactivation and deactivation in sodium channels by comparing ionic flux and charge movement in skeletal muscle channels, using mutations of DIII-DIV linker charges. Charge altering and substituting mutations at K-1317, K-1318 depolarized the g(V) curve but hyperpolarized the h(infinity) curve. Charge reversal and substitution at this locus reduced the apparent voltage sensitivity of open- and closed-state fast inactivation. These effects were not observed with charge reversal at E-1314, E-1315. Mutations swapping or neutralizing the negative cluster at 1314, 1315 and the positive cluster at 1317, 1318 indicated that local interactions dictate the coupling of activation to fast inactivation. Gating charge was immobilized before channel entry into fast inactivation in hNa(V)1.4 but to a lesser extent in mutations at K-1317, K-1318. These results suggest that charge is preferentially immobilized in channels inactivating from the open state. Recovery of gating charge proceeded with a single, fast phase in the double mutation K-1317R, K-1318R. This mutation also partially uncoupled recovery from deactivation. Our findings indicate that charged residues near the fast inactivation "particle" allosterically interact with voltage sensors to control aspects of gating in sodium channels.     

Shaker IR T449 Mutants Separate C- from U-Type Inactivation

Previous studies demonstrated that slow inactivation of the Shaker potassium channel can be made ~100-fold faster or slower by point mutations at a site in the outer pore (T449). However, the discovery that two forms of slow inactivation coexist in Shaker raises the question of which inactivation process is affected by mutation. Equivalent mutations in K_V2.1, a channel exhibiting only U-type inactivation, have minimal effects on inactivation, suggesting that mutation of Shaker T449 acts on C-type inactivation alone, a widely held yet untested hypothesis. This study reexamines mutations at Shaker T449, confirming that T449A speeds inactivation and T449Y/V slow it. T449Y and T449V exhibit U-type inactivation that is enhanced by high extracellular potassium, in contrast to C-type inactivation in T449A which is inhibited by high potassium. Automated parameter estimation for a 12-state Markov model suggests that U-type inactivation occurs mainly from closed states upon weak depolarization, but primarily from the open state at positive voltages. The model also suggests that WT channels, which in this study exhibit mostly C-type inactivation, recover from inactivation through closed-inactivated states, producing voltage-dependent recovery. This suggests that both C-type and U-type inactivation involve both open-inactivated and closed-inactivated states.     

An Intersubunit Salt Bridge near the Selectivity Filter Stabilizes the Active State of Kir1.1

ROMK (Kir1.1) potassium channels are closed by internal acidification with a pKa of 6.7 ± 0.01 in 100 mM external K and a pKa of 7.0 ± 0.01 in 1 mM external K. Internal acidification in 1 mM K (but not 100 mM K) not only closed the pH gate but also inactivated Kir1.1, such that realkalization did not restore channel activity until high K was returned to the bath. We identified a new putative intersubunit salt bridge (R128-E132-Kir1.1b) in the P-loop of the channel near the selectivity filter that affected the K sensitivity of the inactivation process. Mutation of either R128-Kir1.1b or E132-Kir1.1b caused inactivation in both 1 mM and 100 mM external K during oocyte acidification. However, 300 mM external K (but not 200 mM Na + 100 mM K) protected both E132Q and R128Y from inactivation. External application of a modified honey-bee toxin, tertiapin Q (TPNQ), also protected Kir1.1 from inactivation in 1 mM K and protected E132Q and R128Y from inactivation in 100 mM K, which suggests that TPNQ binding to the outer mouth of the channel stabilizes the active state. Pretreatment of Kir1.1 with external Ba prevented Kir1.1 inactivation, similar to pretreatment with TPNQ. In addition, mutations that disrupted transmembrane helix H-bonding (K61M-Kir1.1b) or stabilized a selectivity filter to helix-pore linkage (V121T-Kir1.1b) also protected both E132Q and R128Y from inactivation in 1 mM K and 100 mM K. Our results are consistent with Kir inactivation arising from conformational changes near the selectivity filter, analogous to C-type inactivation.     

Three new familial hemiplegic migraine mutants affect P/Q-type Ca(2+) channel kinetics

Missense mutations in the pore-forming human alpha(1A) subunit of neuronal P/Q-type Ca(2+) channels are associated with familial hemiplegic migraine. We studied the functional consequences on P/Q-type Ca(2+) channel function of three recently identified mutations, R583Q, D715E, and V1457L after introduction into rabbit alpha(1A) and expression in Xenopus laevis oocytes. The potential for half-maximal channel activation of Ba(2+) inward currents was shifted by > 9 mV to more negative potentials in all three mutants. The potential for half-maximal channel inactivation was shifted by > 7 mV in the same direction in R583Q and D715E. Biexponential current inactivation during 3-s test pulses was significantly faster in D715E and slower in V1457L than in wild type. Mutations R583Q and V1457L delayed the time course of recovery from channel inactivation. The decrease of peak current through R583Q (30.2%) and D715E (30. 1%) but not V1457L (18.7%) was more pronounced during 1-Hz trains of 15 100-ms pulses than in wild type (18.2%). Our data demonstrate that the mutations R583Q, D715E, and V1457L, like the previously reported mutations T666M, V714A, and I1819L, affect P/Q-type Ca(2+) channel gating. We therefore propose that altered channel gating represents a common pathophysiological mechanism in familial hemiplegic migraine.     

External nickel blocks human Kv1.5 channels stably expressed in CHO cells

We have investigated the actions of Nickel (Ni²⁺) on a human cardiac potassium channel (hKv1.5), the main component of human atrial ultra-rapid delayed rectifier current, stably expressed in Chinese hamster ovary cell line using the whole-cell voltage-clamp technique. External Ni²⁺ reversibly decreased the amplitude of the current in a concentration-dependent manner. The concentration for half-maximum inhibition of the current at +50 mV was 568 μm. The activation, deactivation, reactivation kinetics of the current were not affected by Ni²⁺. Block was not voltage-dependent but frequency-dependent block was apparent. The extent of channel block during the first pulse increased when the duration of exposure to Ni²⁺, prior to channel activation, was prolonged indicating that Ni²⁺ interacted with hKv1.5 in the closed state. The percentage of current remaining in presence of Ni²⁺ decreased steeply over the range of steady-state channel inactivation, consistent with an enhanced block with increased inactivation. This suggests that Ni²⁺ preferentially blocks nonconducting hKv1.5 channels, either in the resting or inactivated state in a concentration-dependent manner. The data indicate that the mechanisms of hKv1.5 channel inhibition by Ni²⁺ are distinct from those of other K⁺ channels. Received: 12 October 2000/Revised: 14 May 2001     

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.     

KCNK?: opening and closing the 2-P-domain potassium leak channel entails "C-type" gating of the outer pore.

Essential to nerve and muscle function, little is known about how potassium leak channels operate. KCNK? opens and closes in a kinase-dependent fashion. Here, the transition is shown to correspond to changes in the outer aspect of the ion conduction pore. Voltage-gated potassium (VGK) channels open and close via an internal gate; however, they also have an outer pore gate that produces "C-type" inactivation. While KCNK? does not inactivate, KCNK? and VGK channels respond in like manner to outer pore blockers, potassium, mutations, and chemical modifiers. Structural relatedness is confirmed: VGK residues that come close during C-type gating predict KCNK? sites that crosslink (after mutation to cysteine) to yield channels controlled by reduction and oxidization. We conclude that similar outer pore gates mediate KCNK? opening and closing and VGK channel C-type inactivation despite their divergent structures and physiological roles.