N-type Inactivation in the Mammalian Shaker K+ Channel Kv1.4

Mammalian voltage-gated K⁺ channels are oligomeric proteins, some of which may be composed in vivo of subunits derived from several similar genes. We have studied N-type inactivation in the rapidly inactivating Kv1.4 channel and, in specific, heteromultimers of this gene product with Kv1.5 noninactivating subunits. Heteromultimeric channels were analyzed for the stoichiometry of Kv1.4:Kv1.5 subunits by observing shifts in the midpoints of steady-state availability from that of homomultimeric channels. This analysis was employed to examine inactivation of heteromultimeric channels expressed in Xenopus oocytes using two model systems: by expression of a Kv1.4–Kv1.5 tandem fusion construct and by coexpression of native Kv1.4 and Kv1.5 channels across a wide relative concentration range of microinjected mRNA. Additionally, inactivation was examined in coexpression experiments of N-terminal deletion mutants of Kv1.4. We found that (i) a single inactivating subunit conferred inactivation in all hetero-multimers studied; (ii) the rate of inactivation could not be distinguished in channels containing two inactivating subunits from those containing one inactivating subunit; and (iii) large deletions in the linker region between the N-terminal inactivation region and the first membrane-spanning domain had no effect on the rate of inactivation. These data confirm the importance of the proximal N-terminal region in the inactivation of mammalian Kv1.4 channels, and suggest that the inactivation particle remains in close proximity to the permeation pathway even when the channel is in the open state. Received: 24 August 1995/Revised: 7 February 1996     

Structural determinants of Kvbeta1.3-induced channel inactivation: a hairpin modulated by PIP2

Inactivation of voltage-gated Kv1 channels can be altered by Kvbeta subunits, which block the ion-conducting pore to induce a rapid ('N-type') inactivation. Here, we investigate the mechanisms and structural basis of Kvbeta1.3 interaction with the pore domain of Kv1.5 channels. Inactivation induced by Kvbeta1.3 was antagonized by intracellular PIP(2). Mutations of R5 or T6 in Kvbeta1.3 enhanced Kv1.5 inactivation and markedly reduced the effects of PIP(2). R5C or T6C Kvbeta1.3 also exhibited diminished binding of PIP(2) compared with wild-type channels in an in vitro lipid-binding assay. Further, scanning mutagenesis of the N terminus of Kvbeta1.3 revealed that mutations of L2 and A3 eliminated N-type inactivation. Double-mutant cycle analysis indicates that R5 interacts with A501 and T480 of Kv1.5, residues located deep within the pore of the channel. These interactions indicate that Kvbeta1.3, in contrast to Kvbeta1.1, assumes a hairpin structure to inactivate Kv1 channels. Taken together, our findings indicate that inactivation of Kv1.5 is mediated by an equilibrium binding of the N terminus of Kvbeta1.3 between phosphoinositides (PIPs) and the inner pore region of the channel.     

An inactivation gate in the selectivity filter of KCNQ1 potassium channels

Inactivation is an inherent property of most voltage-gated K⁺ channels. While fast N-type inactivation has been analyzed in biophysical and structural details, the mechanisms underlying slow inactivation are yet poorly understood. Here, we characterized a slow inactivation mechanism in various KCNQ1 pore mutants, including L273F, which hinders entry of external Ba²⁺ to its deep site in the pore and traps it by slowing its egress. Kinetic studies, molecular modeling, and dynamics simulations suggest that this slow inactivation involves conformational changes that converge to the outer carbonyl ring of the selectivity filter, where the backbone becomes less flexible. This mechanism involves acceleration of inactivation kinetics and enhancement of Ba²⁺ trapping at elevated external K⁺ concentrations. Hence, KCNQ1 slow inactivation considerably differs from C-type inactivation where vacation of K⁺ from the filter was invoked. We suggest that trapping of K⁺ at s₁ due to filter rigidity and hindrance of the dehydration-resolvation transition underlie the slow inactivation of KCNQ1 pore mutants.     

Differential modulation of Kv1 channel-mediated currents by co-expression of Kvbeta3 subunit in a mammalian cell-line

The effect of Kvbeta3 subunit co-expression on currents mediated by the Shaker-related channels Kv1.1 to Kv1.6 in Chinese hamster ovary (CHO) cells was studied with patch-clamp techniques. In the presence of Kvbeta3, differences in the voltage dependence of activation for Kv1.1, Kv1.3 and Kv1.6 were detected, but not for Kv1.2- and Kv1.4-mediated currents. Co-expression of Kvbeta3 did not cause a significant increase in current density for any of the tested channels. In contrast to previous studies in Xenopus oocyte expression system, Kvbeta3 confered a rapid inactivation to all except Kv1.3 channels. Also, Kv1.6 channels that possess an N-type inactivation prevention (NIP) domain for Kvbeta1.1, inactivated rapidly when co-expressed with Kvbeta3. Onset and recovery kinetics of channel inactivation distinctly differed for the various Kv1alpha/Kvbeta3 subunit combinations investigated in this study. The results indicate that the choice of expression system may critically determine Kvbeta3 inactivating activity. This suggests that the presence of an inactivating domain and a receptor in a channel pore, although necessary, may not be sufficient for an effective rapid N-type inactivation of Kv1 channels in heterologous expression systems.     

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.     

Coupling of voltage-dependent potassium channel inactivation and oxidoreductase active site of Kvbeta subunits

The accessory beta subunits of voltage-dependent potassium (Kv) channels form tetramers arranged with 4-fold rotational symmetry like the membrane-integral and pore-forming alpha subunits (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell. 90, 943-952). The crystal structure of the Kvbeta2 subunit shows that Kvbeta subunits are oxidoreductase enzymes containing an active site composed of conserved catalytic residues, a nicotinamide (NADPH)-cofactor, and a substrate binding site. Also, Kvbeta subunits with an N-terminal inactivating domain like Kvbeta1.1 (Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej, D. N., Dolly, O., and Pongs, O. (1994) Nature 369, 289-294) and Kvbeta3.1 (Heinemann, S. H., Rettig, J., Graack, H. R., and Pongs, O. (1996) J. Physiol. (Lond.) 493, 625-633) confer rapid N-type inactivation to otherwise non-inactivating channels. Here we show by a combination of structural modeling and electrophysiological characterization of structure-based mutations that changes in Kvbeta oxidoreductase activity may markedly influence the gating mode of Kv channels. Amino acid substitutions of the putative catalytic residues in the Kvbeta1.1 oxidoreductase active site attenuate the inactivating activity of Kvbeta1.1 in Xenopus oocytes. Conversely, mutating the substrate binding domain and/or the cofactor binding domain rescues the failure of Kvbeta3.1 to confer rapid inactivation to Kv1.5 channels in Xenopus oocytes. We propose that Kvbeta oxidoreductase activity couples Kv channel inactivation to cellular redox regulation.     

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.     

The Link between Ion Permeation and Inactivation Gating of Kv4 Potassium Channels

Kv4 potassium channels undergo rapid inactivation but do not seem to exhibit the classical N-type and C-type mechanisms present in other Kv channels. We have previously hypothesized that Kv4 channels preferentially inactivate from the preopen closed state, which involves regions of the channel that contribute to the internal vestibule of the pore. To further test this hypothesis, we have examined the effects of permeant ions on gating of three Kv4 channels (Kv4.1, Kv4.2, and Kv4.3) expressed in Xenopus oocytes. Rb⁺ is an excellent tool for this purpose because its prolonged residency time in the pore delays K⁺ channel closing. The data showed that, only when Rb⁺ carried the current, both channel closing and the development of macroscopic inactivation are slowed (1.5- to 4-fold, relative to the K⁺ current). Furthermore, macroscopic Rb⁺ currents were larger than K⁺ currents (1.2- to 3-fold) as the result of a more stable open state, which increases the maximum open probability. These results demonstrate that pore occupancy can influence inactivation gating in a manner that depends on how channel closing impacts inactivation from the preopen closed state. By examining possible changes in ionic selectivity and the influence of elevating the external K⁺ concentration, additional experiments did not support the presence of C-type inactivation in Kv4 channels.     

Inter-Subunit Interactions across the Upper Voltage Sensing-Pore Domain Interface Contribute to the Concerted Pore Opening Transition of Kv Channels

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.     

The epilepsy-linked Lgi1 protein assembles into presynaptic Kv1 channels and inhibits inactivation by Kvbeta1

The voltage-gated potassium (Kv) channel subunit Kv1.1 is a major constituent of presynaptic A-type channels that modulate synaptic transmission in CNS neurons. Here, we show that Kv1.1-containing channels are complexed with Lgi1, the functionally unassigned product of the leucine-rich glioma inactivated gene 1 (LGI1), which is causative for an autosomal dominant form of lateral temporal lobe epilepsy (ADLTE). In the hippocampal formation, both Kv1.1 and Lgi1 are coassembled with Kv1.4 and Kvbeta1 in axonal terminals. In A-type channels composed of these subunits, Lgi1 selectively prevents N-type inactivation mediated by the Kvbeta1 subunit. In contrast, defective Lgi1 molecules identified in ADLTE patients fail to exert this effect resulting in channels with rapid inactivation kinetics. The results establish Lgi1 as a novel subunit of Kv1.1-associated protein complexes and suggest that changes in inactivation gating of presynaptic A-type channels may promote epileptic activity.     

NH2-terminal inactivation peptide binding to C-type-inactivated Kv channels

In many voltage-gated K(+) channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na(+) permeability of C-type-inactivated K(+) channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type-inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na(+) tail currents normally observed through C-type-inactivated channels, an effective blockade of the peak Na(+) tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type-inactivated channels. In C-type-deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type-inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na(+) tail of C-type-inactivated channels. Stable binding between the inactivation peptide and the C-type-inactivated state results in slower current decay, and a reduction of the Na(+) tail current magnitude, due to slower transition of channels through the Na(+)-permeable states traversed during recovery from inactivation.     

Assembly and suppression of endogenous Kv1.3 channels in human T cells

The predominant K+ channel in human T lymphocytes is Kv1.3, which inactivates by a C-type mechanism. To study assembly of these tetrameric channels in Jurkat, a human T-lymphocyte cell line, we have characterized the formation of heterotetrameric channels between endogenous wild-type (WT) Kv1.3 subunits and heterologously expressed mutant (A413V) Kv1.3 subunits. We use a kinetic analysis of C-type inactivation of currents produced by homotetrameric channels and heterotetrameric channels to determine the distribution of channels with different subunit stoichiometries. The distributions are well- described by either a binomial distribution or a binomial distribution plus a fraction of WT homotetramers, indicating that subunit assembly is a random process and that tetramers expressed in the plasma membrane do not dissociate and reassemble. Additionally, endogenous Kv1.3 current is suppressed by a heterologously expressed truncated Kv1.3 that contains the amino terminus and the first two transmembrane segments. The time course for suppression, which is maximal at 48 h after transfection, overlaps with the time interval for heterotetramer formation between heterologously expressed A413V and endogenous WT channels. Our findings suggest that diversity of K+ channel subtypes in a cell is regulated not by spatial segregation of monomeric pools, but rather by the degree of temporal overlap and the kinetics of subunit expression.     

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.     

External pore collapse as an inactivation mechanism for Kv4.3 K+ channels

Kv4 channels are thought to lack a C-type inactivation mechanism (collapse of the external pore) and to inactivate as a result of a concerted action of cytoplasmic regions of the channel. To investigate whether Kv4 channels have outer pore conformational changes during the inactivation process, the inactivation properties of Kv4.3 were characterized in 0 mM and in 2 mM external K+ in whole-cell voltage-clamp experiments. Removal of external K+ increased the inactivation rates and favored cumulative inactivation by repetitive stimulation. The reduction in current amplitude during repetitive stimulation and the faster inactivation rates in 0 mM external K+ were not due to changes in the voltage dependence of channel opening or to internal K+ depletion. The extent of the collapse of the K+ conductance upon removal of external K+ was more pronounced in NMG+-than in Na+-containing solutions. The reduction in the current amplitude during cumulative inactivation by repetitive stimulation is not associated with kinetic changes, suggesting that it is due to a diminished number of functional channels with unchanged gating properties. These observations meet the criteria for a typical C-type inactivation, as removal of external K+ destabilizes the conducting state, leading to the collapse of the pore. A tentative model is presented, in which K+ bound to high-affinity K+-binding sites in the selectivity filter destabilizes an outer neighboring K+ modulatory site that is saturated at approximately 2 mM external K+. We conclude that Kv4 channels have a C-type inactivation mechanism and that previously reported alterations in the inactivation rates after N- and C- termini mutagenesis may arise from secondary changes in the electrostatic interactions between K+-binding sites in the selectivity filter and the neighboring K+-modulatory site, that would result in changes in its K+ occupancy.     

A model of the interaction between N-type and C-type inactivation in Kv1.4 channels

Kv1.4 channels are Shaker-related voltage-gated potassium channels with two distinct inactivation mechanisms. Fast N-type inactivation operates by a ball-and-chain mechanism. Slower C-type inactivation is not so well defined, but involves intracellular and extracellular conformational changes of the channel. We studied the interaction between inactivation mechanisms using two-electrode voltage-clamp of Kv1.4 and Kv1.4ΔN (amino acids 2–146 deleted to remove N-type inactivation) heterologously expressed in Xenopus oocytes. We manipulated C-type inactivation by introducing a lysine-tyrosine point mutation (K532Y, equivalent to Shaker T449Y) that diminishes C-type inactivation. We used experimental data to develop a comprehensive computer model of Kv1.4 channels to determine the interaction between activation and N- and C-type inactivation mechanisms needed to replicate the experimental data. C-type inactivation began at lower voltage preactivated states, whereas N-type inactivation was coupled directly to the open state. A model with distinct N- and C-type inactivated states was not able to reproduce experimental data, and direct transitions between N- and C-type inactivated states were required, i.e., there is coupling between N- and C-type inactivated states. C-type inactivation is the rate-limiting step determining recovery from inactivation, so understanding C-type inactivation, and how it is coupled to N-type inactivation, is critical in understanding how channels act to repetitive stimulation.     

Gating charge immobilization in Kv4.2 channels: the basis of closed-state inactivation

Kv4 channels mediate the somatodendritic A-type K+ current (I(SA)) in neurons. The availability of functional Kv4 channels is dynamically regulated by the membrane potential such that subthreshold depolarizations render Kv4 channels unavailable. The underlying process involves inactivation from closed states along the main activation pathway. Although classical inactivation mechanisms such as N- and P/C-type inactivation have been excluded, a clear understanding of closed-state inactivation in Kv4 channels has remained elusive. This is in part due to the lack of crucial information about the interactions between gating charge (Q) movement, activation, and inactivation. To overcome this limitation, we engineered a charybdotoxin (CTX)-sensitive Kv4.2 channel, which enabled us to obtain the first measurements of Kv4.2 gating currents after blocking K+ conduction with CTX (Dougherty and Covarrubias. 2006J. Gen. Physiol. 128:745-753). Here, we exploited this approach further to investigate the mechanism that links closed-state inactivation to slow Q-immobilization in Kv4 channels. The main observations revealed profound Q-immobilization at steady-state over a range of hyperpolarized voltages (-110 to -75 mV). Depolarization in this range moves <5% of the observable Q associated with activation and is insufficient to open the channels significantly. The kinetics and voltage dependence of Q-immobilization and ionic current inactivation between -153 and -47 mV are similar and independent of the channel's proximal N-terminal region (residues 2-40). A coupled state diagram of closed-state inactivation with a quasi-absorbing inactivated state explained the results from ionic and gating current experiments globally. We conclude that Q-immobilization and closed-state inactivation at hyperpolarized voltages are two manifestations of the same process in Kv4.2 channels, and propose that inactivation in the absence of N- and P/C-type mechanisms involves desensitization to voltage resulting from a slow conformational change of the voltage sensors, which renders the channel's main activation gate reluctant to open.     

Stoichiometry of altered hERG1 channel gating by small molecule activators

Voltage-gated K⁺ channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K⁺ selective conductance. In some K⁺ channels, the selectivity filter can undergo a conformational change to reduce K⁺ flux by a mechanism called C-type inactivation. In human ether-a-go-go–related gene 1 (hERG1) K⁺ channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome.     

N-type Inactivation Features of Kv4.2 Channel Gating

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Δ40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K⁺. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Δ40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Δ40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.     

The electrically silent Kv6.4 subunit confers hyperpolarized gating charge movement in Kv2.1/Kv6.4 heterotetrameric channels

The voltage-gated K(+) (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ~40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K(+) equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits.     

Voltage-dependent C-type inactivation in a constitutively open K+ channel

Most voltage-gated potassium (Kv) channels undergo C-type inactivation during sustained depolarization. The voltage dependence and other mechanistic aspects of this process are debated, and difficult to elucidate because of concomitant voltage-dependent activation. Here, we demonstrate that MinK-KCNQ1 (I_Ks) channels with an S6-domain mutation, F340W in KCNQ1, exhibit constitutive activation but voltage-dependent C-type inactivation. F340W-I_Ks inactivation was sensitive to extracellular cation concentration and species, and it altered ion selectivity, suggestive of pore constriction. The rate and extent of F340W-I_Ks inactivation and recovery from inactivation were voltage-dependent with physiologic intracellular ion concentrations, and in the absence or presence of external K⁺, with an estimated gating charge, z_i, of ∼1. Finally, double-mutant channels with a single S4 charge neutralization (R231A,F340W-I_Ks) exhibited constitutive C-type inactivation. The results suggest that F340W-I_Ks channels exhibit voltage-dependent C-type inactivation involving S4, without the necessity for voltage-dependent opening, allosteric coupling to voltage-dependent S6 transitions occurring during channel opening, or voltage-dependent changes in ion occupancy. The data also identify F340 as a critical hub for KCNQ1 gating processes and their modulation by MinK, and present a unique system for further mechanistic studies of the role of coupling of C-type inactivation to S4 movement, without contamination from voltage-dependent activation.