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

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

S1 constrains S4 in the voltage sensor domain of Kv7.1 K+ channels

Voltage-gated K(+) channels comprise a central pore enclosed by four voltage-sensing domains (VSDs). While movement of the S4 helix is known to couple to channel gate opening and closing, the nature of S4 motion is unclear. Here, we substituted S4 residues of Kv7.1 channels by cysteine and recorded whole-cell mutant channel currents in Xenopus oocytes using the two-electrode voltage-clamp technique. In the closed state, disulfide and metal bridges constrain residue S225 (S4) nearby C136 (S1) within the same VSD. In the open state, two neighboring I227 (S4) are constrained at proximity while residue R228 (S4) is confined close to C136 (S1) of an adjacent VSD. Structural modeling predicts that in the closed to open transition, an axial rotation (approximately 190 degrees) and outward translation of S4 (approximately 12 A) is accompanied by VSD rocking. This large sensor motion changes the intra-VSD S1-S4 interaction to an inter-VSD S1-S4 interaction. These constraints provide a ground for cooperative subunit interactions and suggest a key role of the S1 segment in steering S4 motion during Kv7.1 gating.     

Proline Scan of the hERG Channel S6 Helix Reveals the Location of the Intracellular Pore Gate

In Shaker-like channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline motif, which introduces a kink that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go-related gene (hERG) channels lack the proline-valine-proline motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we show that proline substitutions within the S6 of hERG perturbed pore gate closure, trapping channels in the open state. Performing a proline scan of the inner S6 helix, from Ile⁶⁵⁵ to Tyr⁶⁶⁷ revealed that gate perturbation occurred with proximal (I655P-Q664P), but not distal (R665P-Y667P) substitutions, suggesting that Gln⁶⁶⁴ marks the position of the intracellular gate in hERG channels. Using voltage-clamp fluorimetry and gating current analysis, we demonstrate that proline substitutions trap the activation gate open by disrupting the coupling between the voltage-sensing unit and the pore of the channel. We characterize voltage sensor movement in one such trapped-open mutant channel and demonstrate the kinetics of what we interpret to be intrinsic hERG voltage sensor movement.     

Proline Scan of the hERG Channel S6 Helix Reveals the Location of the Intracellular Pore Gate

In Shaker-like channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline motif, which introduces a kink that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go-related gene (hERG) channels lack the proline-valine-proline motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we show that proline substitutions within the S6 of hERG perturbed pore gate closure, trapping channels in the open state. Performing a proline scan of the inner S6 helix, from Ile⁶⁵⁵ to Tyr⁶⁶⁷ revealed that gate perturbation occurred with proximal (I655P-Q664P), but not distal (R665P-Y667P) substitutions, suggesting that Gln⁶⁶⁴ marks the position of the intracellular gate in hERG channels. Using voltage-clamp fluorimetry and gating current analysis, we demonstrate that proline substitutions trap the activation gate open by disrupting the coupling between the voltage-sensing unit and the pore of the channel. We characterize voltage sensor movement in one such trapped-open mutant channel and demonstrate the kinetics of what we interpret to be intrinsic hERG voltage sensor movement.     

Functional interactions between residues in the S1, S4, and S5 domains of Kv2.1

The voltage-gated potassium channel subunit Kv2.1 forms heterotetrameric channels with the silent subunit Kv6.4. Chimeric Kv2.1 channels containing a single transmembrane segment from Kv6.4 have been shown to be functional. However, a Kv2.1 chimera containing both S1 and S5 from Kv6.4 was not functional. Back mutation of individual residues in this chimera (to the Kv2.1 counterpart) identified four positions that were critical for functionality: A200V and A203T in S1, and T343M and P347S in S5. To test for possible interactions in Kv2.1, we used substitutions with charged residues and tryptophan for the outermost pair 203/347. Combinations of substitutions with opposite charges at both T203 and S347 were tolerated but resulted in channels with altered gating kinetics, as did the combination of negatively charged aspartate substitutions. Double mutant cycle analysis with these mutants indicated that both residues are energetically coupled. In contrast, replacing both residues with a positively charged lysine together (T203K + S347K) was not tolerated and resulted in a folding or trafficking deficiency. The nonfunctionality of the T203K + S347K mutation could be restored by introducing the R300E mutation in the S4 segment of the voltage sensor. These results indicate that these specific S1, S4, and S5 residues are in close proximity and interact with each other in the functional channel, but are also important determinants for Kv2.1 channel maturation. These data support the view of an anchoring interaction between S1 and S5, but indicate that this interaction surface is more extensive than previously proposed.     

Voltage-dependent gating rearrangements in the intracellular T1-T1 interface of a K+ channel

The intracellular tetramerization domain (T1) of most eukaryotic voltage-gated potassium channels (Kv channels) exists as a "hanging gondola" below the transmembrane regions that directly control activation gating via the electromechanical coupling between the S4 voltage sensor and the main S6 gate. However, much less is known about the putative contribution of the T1 domain to Kv channel gating. This possibility is mechanistically intriguing because the T1-S1 linker connects the T1 domain to the voltage-sensing domain. Previously, we demonstrated that thiol-specific reagents inhibit Kv4.1 channels by reacting in a state-dependent manner with native Zn(2+) site thiolate groups in the T1-T1 interface; therefore, we concluded that the T1-T1 interface is functionally active and not protected by Zn(2+) (Wang, G., M. Shahidullah, C.A. Rocha, C. Strang, P.J. Pfaffinger, and M. Covarrubias. 2005. J. Gen. Physiol. 126:55-69). Here, we co-expressed Kv4.1 channels and auxiliary subunits (KChIP-1 and DPPX-S) to investigate the state and voltage dependence of the accessibility of MTSET to the three interfacial cysteines in the T1 domain. The results showed that the average MTSET modification rate constant (k(MTSET)) is dramatically enhanced in the activated state relative to the resting and inactivated states (approximately 260- and approximately 47-fold, respectively). Crucially, under three separate conditions that produce distinct activation profiles, k(MTSET) is steeply voltage dependent in a manner that is precisely correlated with the peak conductance-voltage relations. These observations strongly suggest that Kv4 channel gating is tightly coupled to voltage-dependent accessibility changes of native T1 cysteines in the intersubunit Zn(2+) site. Furthermore, cross-linking of cysteine pairs across the T1-T1 interface induced substantial inhibition of the channel, which supports the functionally dynamic role of T1 in channel gating. Therefore, we conclude that the complex voltage-dependent gating rearrangements of eukaryotic Kv channels are not limited to the membrane-spanning core but must include the intracellular T1-T1 interface. Oxidative stress in excitable tissues may perturb this interface to modulate Kv4 channel function.     

Molecular coupling in the human ether-a-go-go-related gene-1 (hERG1) K+ channel inactivation pathway

Emerging evidence suggests that K(+) channel inactivation involves coupling between residues in adjacent regions of the channel. Human ether-a-go-go-related gene-1 (hERG1) K(+) channels undergo a fast inactivation gating process that is crucial for maintaining electrical stability in the heart. The molecular mechanisms that drive inactivation in hERG1 channels are unknown. Using alanine scanning mutagenesis, we show that a pore helix residue (Thr-618) that points toward the S5 segment is critical for normal inactivation gating. Amino acid substitutions at position 618 modulate the free energy of inactivation gating, causing enhanced or reduced inactivation. Mutation of an S5 residue that is predicted to be adjacent to Thr-618 (W568L) abolishes inactivation and alters ion selectivity. The introduction of the Thr-618-equivalent residue in Kv1.5 enhances inactivation. Molecular dynamic simulations of the Kv1.2 tetramer reveal van der Waals coupling between hERG1 618- and 568-equivalent residues and a significant increase in interaction energies when threonine is introduced at the 618-equivalent position. We propose that coupling between the S5 segment and pore helix may participate in the inactivation process in hERG1 channels.     

KCNQ1 Channels Do Not Undergo Concerted but Sequential Gating Transitions in Both the Absence and the Presence of KCNE1 Protein

The co-assembly of KCNQ1 with KCNE1 produces I_KS, a K⁺ current, crucial for the repolarization of the cardiac action potential. Mutations in these channel subunits lead to life-threatening cardiac arrhythmias. However, very little is known about the gating mechanisms underlying KCNQ1 channel activation. Shaker channels have provided a powerful tool to establish the basic gating mechanisms of voltage-dependent K⁺ channels, implying prior independent movement of all four voltage sensor domains (VSDs) followed by channel opening via a last concerted cooperative transition. To determine the nature of KCNQ1 channel gating, we performed a thermodynamic mutant cycle analysis by constructing a concatenated tetrameric KCNQ1 channel and by introducing separately a gain and a loss of function mutation, R231W and R243W, respectively, into the S4 helix of the VSD of one, two, three, and four subunits. The R231W mutation destabilizes channel closure and produces constitutively open channels, whereas the R243W mutation disrupts channel opening solely in the presence of KCNE1 by right-shifting the voltage dependence of activation. The linearity of the relationship between the shift in the voltage dependence of activation and the number of mutated subunits points to an independence of VSD movements, with each subunit incrementally contributing to channel gating. Contrary to Shaker channels, our work indicates that KCNQ1 channels do not experience a late cooperative concerted opening transition. Our data suggest that KCNQ1 channels in both the absence and the presence of KCNE1 undergo sequential gating transitions leading to channel opening even before all VSDs have moved.     

Capturing distinct KCNQ2 channel resting states by metal ion bridges in the voltage-sensor domain

Although crystal structures of various voltage-gated K⁺ (Kv) and Na⁺ channels have provided substantial information on the activated conformation of the voltage-sensing domain (VSD), the topology of the VSD in its resting conformation remains highly debated. Numerous studies have investigated the VSD resting state in the Kv Shaker channel; however, few studies have explored this issue in other Kv channels. Here, we investigated the VSD resting state of KCNQ2, a K⁺ channel subunit belonging to the KCNQ (Kv7) subfamily of Kv channels. KCNQ2 can coassemble with the KCNQ3 subunit to mediate the I_M current that regulates neuronal excitability. In humans, mutations in KCNQ2 are associated with benign neonatal forms of epilepsy or with severe epileptic encephalopathy. We introduced cysteine mutations into the S4 transmembrane segment of the KCNQ2 VSD and determined that external application of Cd²⁺ profoundly reduced the current amplitude of S4 cysteine mutants S195C, R198C, and R201C. Based on reactivity with the externally accessible endogenous cysteine C106 in S1, we infer that each of the above S4 cysteine mutants forms Cd²⁺ bridges to stabilize a channel closed state. Disulfide bonds and metal bridges constrain the S4 residues S195, R198, and R201 near C106 in S1 in the resting state, and experiments using concatenated tetrameric constructs indicate that this occurs within the same VSD. KCNQ2 structural models suggest that three distinct resting channel states have been captured by the formation of different S4–S1 Cd²⁺ bridges. Collectively, this work reveals that residue C106 in S1 can be very close to several N-terminal S4 residues for stabilizing different KCNQ2 resting conformations.     

Voltage-dependent gating of hyperpolarization-activated, cyclic nucleotide-gated pacemaker channels: molecular coupling between the S4-S5 and C-linkers

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have a transmembrane topology that is highly similar to voltage-gated K(+) channels, yet HCN channels open in response to membrane hyperpolarization instead of depolarization. The structural basis for the "inverted" voltage dependence of HCN gating and how voltage sensing by the S1-S4 domains is coupled to the opening of the intracellular gate formed by the S6 domain are unknown. Coupling could arise from interaction between specific residues or entire transmembrane domains. We previously reported that the mutation of specific residues in the S4-S5 linker of HCN2 (i.e. Tyr-331 and Arg-339) prevented normal channel closure presumably by disruption of a crucial interaction with the activation gate. Here we hypothesized that the C-linker, a carboxyl terminus segment that connects S6 to the cyclic nucleotide binding domain, interacts with specific residues of the S4-S5 linker to mediate coupling. The recently solved structure of the C-linker of HCN2 indicates that an alpha-helix (the A'-helix) is located near the end of each S6 domain, the presumed location of the activation gate. Ala-scanning mutagenesis of the end of S6 and the A'-helix identified five residues that were important for normal gating as mutations disrupted channel closure. However, partial deletion of the C-linker indicated that the presence of only two of these residues was required for normal coupling. Further mutation analyses suggested that a specific electrostatic interaction between Arg-339 of the S4-S5 linker and Asp-443 of the C-linker stabilizes the closed state and thus participates in the coupling of voltage sensing and activation gating in HCN channels.     

The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating

Human ether-à-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.     

Conserved residues within the putative S4-S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels

The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.     

Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating

The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.     

The S4-S5 linker directly couples voltage sensor movement to the activation gate in the human ether-a'-go-go-related gene (hERG) K+ channel

A key unresolved question regarding the basic function of voltage-gated ion channels is how movement of the voltage sensor is coupled to channel opening. We previously proposed that the S4-S5 linker couples voltage sensor movement to the S6 domain in the human ether-a'-go-go-related gene (hERG) K+ channel. The recently solved crystal structure of the voltage-gated Kv1.2 channel reveals that the S4-S5 linker is the structural link between the voltage sensing and pore domains. In this study, we used chimeras constructed from hERG and ether-a'-go-go (EAG) channels to identify interactions between residues in the S4-S5 linker and S6 domain that were critical for stabilizing the channel in a closed state. To verify the spatial proximity of these regions, we introduced cysteines in the S4-S5 linker and at the C-terminal end of the S6 domain and then probed for the effect of oxidation. The D540C-L666C channel current decreased in an oxidizing environment in a state-dependent manner consistent with formation of a disulfide bond that locked the channel in a closed state. Disulfide bond formation also restricted movement of the voltage sensor, as measured by gating currents. Taken together, these data confirm that the S4-S5 linker directly couples voltage sensor movement to the activation gate. Moreover, rather than functioning simply as a mechanical lever, these findings imply that specific interactions between the S4-S5 linker and the activation gate stabilize the closed channel conformation.     

Double mutant cycle analysis identified a critical leucine residue in the IIS4S5 linker for the activation of the Ca(V)2.3 calcium channel

Mutations in distal S6 were shown to significantly alter the stability of the open state of Ca(V)2.3 (Raybaud, A., Baspinar, E. E., Dionne, F., Dodier, Y., Sauvé, R., and Parent, L. (2007) J. Biol. Chem. 282, 27944-27952). By analogy with K(V) channels, we tested the hypothesis that channel activation involves electromechanical coupling between S6 and the S4S5 linker in Ca(V)2.3. Among the 11 positions tested in the S4S5 linker of domain II, mutations of the leucine residue at position 596 were found to destabilize significantly the closed state with a -50 mV shift in the activation potential and a -20 mV shift in its charge-voltage relationship as compared with Ca(V)2.3 wt. A double mutant cycle analysis was performed by introducing pairs of glycine residues between S4S5 and S6 of Domain II. Strong coupling energies (ΔΔG(interact) > 2 kcal mol(-1)) were measured for the activation gating of 12 of 39 pairs of mutants. Leu-596 (IIS4S5) was strongly coupled with distal residues in IIS6 from Leu-699 to Asp-704. In particular, the double mutant L596G/I701G showed strong cooperativity with a ΔΔG(interact) ≈6 kcal mol(-1) suggesting that both positions contribute to the activation gating of the channel. Altogether, our results highlight the role of a leucine residue in S4S5 and provide the first series of evidence that the IIS4S5 and IIS6 regions are energetically coupled during the activation of a voltage-gated Ca(V) channel.     

Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action

Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K⁺ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.     

The role of S4 charges in voltage-dependent and voltage-independent KCNQ1 potassium channel complexes

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 alpha subunits to generate the voltage-dependent I(Ks) channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium "leak" currents in gastrointestinal epithelia. Other Kv alpha subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2.     

Coupling between the voltage-sensing and pore domains in a voltage-gated potassium channel

Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4-S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4-S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.     

Movement of the S4 segment in the hERG potassium channel during membrane depolarization

Abstract

The hERG potassium channel is a member of the voltage gated potassium (Kv) channel family, comprising a pore domain and four voltage sensing domains (VSDs). Like other Kv channels, the VSD senses changes in membrane voltage and transmits the signal to gates located in the pore domain; the gates open at positive potentials (activation) and close at negative potentials, thereby controlling the ion flux. hERG, however, differs from other Kv channels in that it is activated slowly but inactivated rapidly – a property that is crucial for the role it plays in the repolarization of the cardiac action potential. Voltage-gating requires movement of gating charges across the membrane electric field, which is accomplished by the transmembrane movement of the fourth transmembrane segment, S4, of the VSD containing the positively charged arginine or lysine residues. Here we ask if the functional differences between hERG and other Kv channels could arise from differences in the transmembrane movement of S4. To address this, we have introduced single cysteine residues into the S4 region of the VSD, expressed the mutant channels in Xenopus oocytes and examined the effect of membrane impermeable para-chloromercuribenzene sulphonate on function by the two-electrode voltage clamp technique. Our results show that depolarization results in the accessibility of seven consecutive S4 residues, including the first two charged residues, K525 and R528, to extracellularly applied reagent. These data indicate that the extent of S4 movement in hERG is similar to other Kv channels, including the archabacterial KvAP and the Shaker channel of Drosophila.     

Demonstration of physical proximity between the N terminus and the S4-S5 linker of the human ether-a-go-go-related gene (hERG) potassium channel

Potassium channels encoded by the human ether-à-go-go-related gene (hERG) contribute to cardiac repolarization as a result of their characteristic gating properties. The hERG channel N terminus acts as a crucial determinant in gating. It is also known that the S4-S5 linker couples the voltage-sensing machinery to the channel gate. Moreover, this linker has been repeatedly proposed as an interaction site for the distal portion of the N terminus controlling channel gating, but direct evidence for such an interaction is still lacking. In this study, we used disulfide bond formation between pairs of engineered cysteines to demonstrate the close proximity between the beginning of the N terminus and the S4-S5 linker. Currents from channels with introduced cysteines were rapidly and strongly attenuated by an oxidizing agent, this effect being maximal for cysteine pairs located around amino acids 3 and 542 of the hERG sequence. The state-dependent modification of the double-mutant channels, but not the single-cysteine mutants, and the ability to readily reverse modification with the reducing agent dithiothreitol indicate that a disulfide bond is formed under oxidizing conditions, locking the channels in a non-conducting state. We conclude that physical interactions between the N-terminal-most segment of the N terminus and the S4-S5 linker constitute an essential component of the hERG gating machinery, thus providing a molecular basis for previous data and indicating an important contribution of these cytoplasmic domains in controlling its unusual gating and hence determining its physiological role in setting the electrical behavior of cardiac and other cell types.