Computer simulation of the KvAP voltage-gated potassium channel: steered molecular dynamics of the voltage sensor

The recent crystal structures of the voltage-gated potassium channel KvAP and its isolated voltage-sensing 'paddle' (composed of segments S1-S4) challenge existing models of voltage gating and raise a number of questions about the structure of the physiologically relevant state. We investigate a possible gating mechanism based on the crystal structures in a 10 ns steered molecular dynamics simulation of KvAP in a membrane-mimetic octane layer. The structure of the full KvAP protein has been modified by restraining the S2-S4 domain to the conformation of the isolated high-resolution paddle structure. After an initial relaxation, the paddle tips are pulled through the membrane from the intracellular to the extracellular side, corresponding to a putative change from closed to open. We describe the effect of this large-scale motion on the central pore domain, which remains largely unchanged, on the protein hydrogen-bonding network and on solvent. We analyze the motion of the S3b-S4 portion of the protein and propose a possible coupling mechanism between the paddle motion and the opening of the channel. Interactions between the arginine residues in S4, solvent and chloride ions are likely to play a role in the gating charge.     

Changes in local S4 environment provide a voltage-sensing mechanism for mammalian hyperpolarization-activated HCN channels

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (approximately 3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.     

Microscopic origin of gating current fluctuations in a potassium channel voltage sensor

Voltage-dependent ion channels open and close in response to changes in membrane electrical potential due to the motion of their voltage-sensing domains (VSDs). VSD charge displacements within the membrane electric field are observed in electrophysiology experiments as gating currents preceding ionic conduction. The elementary charge motions that give rise to the gating current cannot be observed directly, but appear as discrete current pulses that generate fluctuations in gating current measurements. Here we report direct observation of gating-charge displacements in an atomistic molecular dynamics simulation of the isolated VSD from the KvAP channel in a hydrated lipid bilayer on the timescale (10-μs) expected for elementary gating charge transitions. The results reveal that gating-charge displacements are associated with the water-catalyzed rearrangement of salt bridges between the S4 arginines and a set of conserved acidic side chains on the S1-S3 transmembrane segments in the hydrated interior of the VSD.     

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

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

Molecular coupling of S4 to a K(+) channel's slow inactivation gate

The mechanism by which physiological signals regulate the conformation of molecular gates that open and close ion channels is poorly understood. Voltage clamp fluorometry was used to ask how the voltage-sensing S4 transmembrane domain is coupled to the slow inactivation gate in the pore domain of the Shaker K(+) channel. Fluorophores attached at several sites in S4 indicate that the voltage-sensing rearrangements are followed by an additional inactivation motion. Fluorophores attached at the perimeter of the pore domain indicate that the inactivation rearrangement projects from the selectivity filter out to the interface with the voltage-sensing domain. Some of the pore domain sites also sense activation, and this appears to be due to a direct interaction with S4 based on the finding that S4 comes into close enough proximity to the pore domain for a pore mutation to alter the nanoenvironment of an S4-attached fluorophore. We propose that activation produces an S4-pore domain interaction that disrupts a bond between the S4 contact site on the pore domain and the outer end of S6. Our results indicate that this bond holds the slow inactivation gate open and, therefore, we propose that this S4-induced bond disruption triggers inactivation.     

Electrostatic interactions between transmembrane segments mediate folding of Shaker K+ channel subunits.

In voltage-dependent Shaker K+ channels, charged residues E293 in transmembrane segment S2 and R365, R368, and R371 in S4 contribute significantly to the gating charge movement that accompanies activation. Using an intragenic suppression strategy, we have now probed for structural interaction between transmembrane segments S2, S3, and S4 in Shaker channels. Charge reversal mutations of E283 in S2 and K374 in S4 disrupt maturation of the protein. Maturation was specifically and efficiently rescued by second-site charge reversal mutations, indicating that electrostatic interactions exist between E283 in S2 and R368 and R371 in S4, and between K374 in S4 and E293 in S2 and D316 in S3. Rescued subunits were incorporated into functional channels, demonstrating that a native structure was restored. Our data indicate that K374 interacts with E293 and D316 within the same subunit. These electrostatic interactions mediate the proper folding of the protein and are likely to persist in the native structure. Our results raise the possibility that the S4 segment is tilted relative to S2 and S3 in the voltage-sensing domain of Shaker channels. Such an arrangement might provide solvent access to voltage-sensing residues, which we find to be highly tolerant of mutations.     

alpha-helical structural elements within the voltage-sensing domains of a K(+) channel

Voltage-gated K(+) channels are tetramers with each subunit containing six (S1-S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5-S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1-S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K(+) channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of alpha-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting alpha-helical secondary structure. In addition, both the S1-S2 and S3-S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.     

Protein Rearrangements Underlying Slow Inactivation of the Shaker K+ Channel

Voltage-dependent ion channels transduce changes in the membrane electric field into protein rearrangements that gate their transmembrane ion permeation pathways. While certain molecular elements of the voltage sensor and gates have been identified, little is known about either the nature of their conformational rearrangements or about how the voltage sensor is coupled to the gates. We used voltage clamp fluorometry to examine the voltage sensor (S4) and pore region (P-region) protein motions that underlie the slow inactivation of the Shaker K⁺ channel. Fluorescent probes in both the P-region and S4 changed emission intensity in parallel with the onset and recovery of slow inactivation, indicative of local protein rearrangements in this gating process. Two sequential rearrangements were observed, with channels first entering the P-type, and then the C-type inactivated state. These forms of inactivation appear to be mediated by a single gate, with P-type inactivation closing the gate and C-type inactivation stabilizing the gate''s closed conformation. Such a stabilization was due, at least in part, to a slow rearrangement around S4 that stabilizes S4 in its activated transmembrane position. The fluorescence reports of S4 and P-region fluorophore are consistent with an increased interaction of the voltage sensor and inactivation gate upon gate closure, offering insight into how the voltage-sensing apparatus is coupled to a channel gate.     

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

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

Binding site in eag voltage sensor accommodates a variety of ions and is accessible in closed channel

In ether-a-go-go K+ channels, voltage-dependent activation is modulated by ion binding to a site located in an extracellular-facing crevice between transmembrane segments S2 and S3 in the voltage sensor. We find that acidic residues D278 in S2 and D327 in S3 are able to coordinate a variety of divalent cations, including Mg2+, Mn2+, and Ni2+, which have qualitatively similar functional effects, but different half-maximal effective concentrations. Our data indicate that ions binding to individual voltage sensors in the tetrameric channel act without cooperativity to modulate activation gating. We have taken advantage of the unique phenotype of Ni2+ in the D274A channel, which contains a mutation of a nonbinding site residue, to demonstrate that ions can access the binding site from the extracellular solution when the voltage sensor is in the resting conformation. Our results are difficult to reconcile with the x-ray structure of the KvAP K+ channel, in which the binding site residues are widely separated, and with the hydrophobic paddle model for voltage-dependent activation, in which the voltage sensor domain, including the S3-S4 loop, is near the cytoplasmic side of the membrane in the closed channel.     

High-Resolution Orientation and Depth of Insertion of the Voltage-Sensing S4 Helix of a Potassium Channel in Lipid Bilayers

The opening and closing of voltage-gated potassium (Kv) channels are controlled by several conserved Arg residues in the S4 helix of the voltage-sensing domain. The interaction of these positively charged Arg residues with the lipid membrane has been of intense interest for understanding how membrane proteins fold to allow charged residues to insert into lipid bilayers against free-energy barriers. Using solid-state NMR, we have now determined the orientation and insertion depth of the S4 peptide of the KvAP channel in lipid bilayers. Two-dimensional ¹⁵N correlation experiments of macroscopically oriented S4 peptide in phospholipid bilayers revealed a tilt angle of 40° and two possible rotation angles differing by 180° around the helix axis. Remarkably, the tilt angle and one of the two rotation angles are identical to those of the S4 helix in the intact voltage-sensing domain, suggesting that interactions between the S4 segment and other helices of the voltage-sensing domain are not essential for the membrane topology of the S4 helix. ¹³C-³¹P distances between the S4 backbone and the lipid ³¹P indicate a ∼ 9 Å local thinning and 2 Å average thinning of the DMPC (1,2-dimyristoyl-sn-glycero-3-phosphochloline)/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) bilayer, consistent with neutron diffraction data. Moreover, a short distance of 4.6 Å from the guanidinium C^ζ of the second Arg to ³¹P indicates the existence of guanidinium phosphate hydrogen bonding and salt bridges. These data suggest that the structure of the Kv gating helix is mainly determined by protein-lipid interactions instead of interhelical protein-protein interactions, and the S4 amino acid sequence encodes sufficient information for the membrane topology of this crucial gating helix.     

Gating charges in the activation and inactivation processes of the HERG channel

The hERG channel has a relatively slow activation process but an extremely fast and voltage-sensitive inactivation process. Direct measurement of hERG's gating current (Piper, D.R., A. Varghese, M.C. Sanguinetti, and M. Tristani-Firouzi. 2003. PNAS. 100:10534-10539) reveals two kinetic components of gating charge transfer that may originate from two channel domains. This study is designed to address three questions: (1) which of the six positive charges in hERG's major voltage sensor, S4, are responsible for gating charge transfer during activation, (2) whether a negative charge in the cytoplasmic half of S2 (D466) also contributes to gating charge transfer, and (3) whether S4 serves as the sole voltage sensor for hERG inactivation. We individually mutate S4's positive charges and D466 to cysteine, and examine (a) effects of mutations on the number of equivalent gating charges transferred during activation (z(a)) and inactivation (z(i)), and (b) sidedness and state dependence of accessibility of introduced cysteine side chains to a membrane-impermeable thiol-modifying reagent (MTSET). Neutralizing the outer three positive charges in S4 and D466 in S2 reduces z(a), and cysteine side chains introduced into these positions experience state-dependent changes in MTSET accessibility. On the other hand, neutralizing the inner three positive charges in S4 does not affect z(a). None of the charge mutations affect z(i). We propose that the scheme of gating charge transfer during hERG's activation process is similar to that described for the Shaker channel, although hERG has less gating charge in its S4 than in Shaker. Furthermore, channel domain other than S4 contributes to gating charge involved in hERG's inactivation process.     

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.     

Probing the structural basis for differential KCNQ1 modulation by KCNE1 and KCNE2

KCNE1 associates with KCNQ1 to increase its current amplitude and slow the activation gating process, creating the slow delayed rectifier channel that functions as a “repolarization reserve” in human heart. The transmembrane domain (TMD) of KCNE1 plays a key role in modulating KCNQ1 pore conductance and gating kinetics, and the extracellular juxtamembrane (EJM) region plays a modulatory role by interacting with the extracellular surface of KCNQ1. KCNE2 is also expressed in human heart and can associate with KCNQ1 to suppress its current amplitude and slow the deactivation gating process. KCNE1 and KCNE2 share the transmembrane topology and a high degree of sequence homology in TMD and surrounding regions. The structural basis for their distinctly different effects on KCNQ1 is not clear. To address this question, we apply cysteine (Cys) scanning mutagenesis to TMDs and EJMs of KCNE1 and KCNE2. We analyze the patterns of functional perturbation to identify high impact positions, and probe disulfide formation between engineered Cys side chains on KCNE subunits and native Cys on KCNQ1. We also use methanethiosulfonate reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Our data suggest that the TMDs of both KCNE subunits are at about the same location but interact differently with KCNQ1. In particular, the much closer contact of KCNE2 TMD with KCNQ1, relative to that of KCNE1, is expected to impact the allosteric modulation of KCNQ1 pore conductance and may explain their differential effects on the KCNQ1 current amplitude. KCNE1 and KCNE2 also differ in the relationship between their EJMs and KCNQ1. Although the EJM of KCNE1 makes intimate contacts with KCNQ1, there appears to be a crevice between KCNQ1 and KCNE2. This putative crevice may perturb the electrical field around the voltage-sensing domain of KCNQ1, contributing to the differential effects of KCNE2 versus KCNE1 on KCNQ1 gating kinetics.     

Resin-acid derivatives bind to multiple sites on the voltage-sensor domain of the Shaker potassium channel

Voltage-gated potassium (K_V) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker K_V channel increased resin-acid–induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker K_V channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid–binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1–S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner.     

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

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

Molecular coupling between voltage sensor and pore opening in the Arabidopsis inward rectifier K+ channel KAT1

Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open.     

Coupling between voltage sensors and activation gate in voltage-gated K+ channels

Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1-S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5-S6, equivalent to M1-M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4-S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.     

Linker flexibility of IVS3-S4 loops modulates voltage-dependent activation of L-type Ca2+ channels

Extracellular S3-S4 linkers of domain IV (IVS3-S4) of L-type Ca²⁺ channels (Ca_V1) are subject to alternative splicing, resulting into distinct gating profiles serving for diverse physiological roles. However, it has remained elusive what would be the determining factor of IVS3-S4 effects on Ca_V1 channels. In this study, we systematically compared IVS3-S4 variants from Ca_V1.1-1.4, and discover that the flexibility of the linker plays a prominent role in gating characteristics. Chimeric analysis and mutagenesis demonstrated that changes in half activation voltage (V_1/2) or activation time constant (τ) are positively correlated with the numbers of flexible glycine residues within the linker. Moreover, antibodies that reduce IVS3-S4 flexibility negatively shifted V_1/2, emerging as a new category of Ca_V1 enhancers. In summary, our results suggest that the flexibility or rigidity of IVS3-S4 linker underlies its modulations on Ca_V1 activation (V_1/2 and τ), paving the way to dissect the core mechanisms and to develop innovative perturbations pertaining to voltage-sensing S4 and its vicinities.     

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.