Insight into the mechanism of inactivation and pH sensitivity in potassium channels from molecular dynamics simulations

Potassium (K (+)) channels can regulate ionic conduction through their pore by a mechanism, involving the selectivity filter, known as C-type inactivation. This process is rapid in the hERG K (+) channel and is fundamental to its physiological role. Although mutations within hERG are known to remove this process, a structural basis for the inactivation mechanism has yet to be characterized. Using MD simulations based on homology modeling, we observe that the carbonyl of the filter aromatic, Phe627, forming the S 0 K (+) binding site, swiftly rotates away from the conduction axis in the wild-type channel. In contrast, in well-characterized non-inactivating mutant channels, this conformational change occurs less frequently. In the non-inactivating channels, interactions with a water molecule located behind the selectivity filter are critical to the enhanced stability of the conducting state. We observe comparable conformational changes in the acid sensitive TASK-1 channel and propose a common mechanism in these channels for regulating efflux of K (+) ions through the selectivity filter.     

Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA

Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K(+) channel KcsA under near-physiological conditions, using solution-state NMR. In a series of (1)H(15)N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K(+) channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a "pH-sensor" for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed "dual gating" properties of the K(+) channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.     

Voltage-dependent inactivation gating at the selectivity filter of the MthK K+ channel

Voltage-dependent K(+) channels can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel's selectivity filter. C-type inactivation may involve movements of transmembrane voltage sensor domains, although the mechanisms underlying this form of inactivation may be heterogeneous and are often unclear. Here, we report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K(+) channel that lacks a canonical voltage sensor and may thus provide a reduced system to inform on mechanism. In single-channel recordings, we observe that Po decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca(2+) or Ba(2+), suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K(+)] (47 mV per 10-fold increase in [K(+)]), suggesting that K(+) binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K(+) ≈ Rb(+) > Cs(+) > Na(+) > Li(+) ≈ NMG(+). Selectivity of the stabilizing site is weaker than that of sites that determine permeability of these ions, suggesting that the site may lie toward the external end of the MthK selectivity filter. We could describe MthK gating over a wide range of positive voltages and external [K(+)] using kinetic schemes in which the open-conductive state is stabilized by K(+) binding to a site that is not deep within the electric field, with the voltage dependence of inactivation arising from both voltage-dependent K(+) dissociation and transitions between nonconducting (inactivated) states. These results provide a quantitative working hypothesis for voltage-dependent, K(+)-sensitive inactivation gating, a property that may be common to other K(+) channels.     

Cooperative interactions between R531 and acidic residues in the voltage sensing module of hERG1 channels.

HERG1 K(+) channels are critical for modulating the duration of the cardiac action potential. The role of hERG1 channels in maintaining electrical stability in the heart derives from their unusual gating properties: slow activation and fast inactivation. HERG1 channel inactivation is intrinsically voltage sensitive and is not coupled to activation in the same way as in the Shaker family of K(+) channels. We recently proposed that the S4 transmembrane domain functions as the primary voltage sensor for hERG1 activation and inactivation and that distinct regions of S4 contribute to each gating process. In this study, we tested the hypothesis that S4 rearrangements underlying activation and inactivation gating may be associated with distinct cooperative interactions between a key residue in the S4 domain (R531) and acidic residues in neighboring regions (S1 - S3 domains) of the voltage sensing module. Using double-mutant cycle analysis, we found that R531 was energetically coupled to all acidic residues in S1-S3 during activation, but was coupled only to acidic residues near the extracellular portion of S2 and S3 (D456, D460 and D509) during inactivation. We propose that hERG1 activation involves a cooperative conformational change involving the entire voltage sensing module, while inactivation may involve a more limited interaction between R531 and D456, D460 and D509.     

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.     

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.     

The pore domain outer helix contributes to both activation and inactivation of the HERG K+ channel

Ion flow in many voltage-gated K(+) channels (VGK), including the (human ether-a-go-go-related gene) hERG channel, is regulated by reversible collapse of the selectivity filter. hERG channels, however, exhibit low sequence homology to other VGKs, particularly in the outer pore helix (S5) domain, and we hypothesize that this contributes to the unique activation and inactivation kinetics in hERG K(+) channels that are so important for cardiac electrical activity. The S5 domain in hERG identified by NMR spectroscopy closely corresponded to the segment predicted by bioinformatics analysis of 676 members of the VGK superfamily. Mutations to approximately every third residue, from Phe(551) to Trp(563), affected steady state activation, whereas mutations to approximately every third residue on an adjacent face and spanning the entire S5 segment perturbed inactivation, suggesting that the whole span of S5 experiences a rearrangement associated with inactivation. We refined a homology model of the hERG pore domain using constraints from the mutagenesis data with residues affecting inactivation pointing in toward S6. In this model the three residues with maximum impact on activation (W563A, F559A, and F551A) face out toward the voltage sensor. In addition, the residues that when mutated to alanine, or from alanine to valine, that did not express (Ala(561), His(562), Ala(565), Trp(568), and Ile(571)), all point toward the pore helix and contribute to close hydrophobic packing in this region of the channel.     

H bonding at the helix-bundle crossing controls gating in Kir potassium channels

Specific stimuli such as intracellular H+ and phosphoinositides (e.g., PIP2) gate inwardly rectifying potassium (Kir) channels by controlling the reversible transition between the closed and open states. This gating mechanism underlies many aspects of Kir channel physiology and pathophysiology; however, its structural basis is not well understood. Here, we demonstrate that H+ and PIP2 use a conserved gating mechanism defined by similar structural changes in the transmembrane (TM) helices and the selectivity filter. Our data support a model in which the gating motion of the TM helices is controlled by an intrasubunit hydrogen bond between TM1 and TM2 at the helix-bundle crossing, and we show that this defines a common gating motif in the Kir channel superfamily. Furthermore, we show that this proposed H-bonding interaction determines Kir channel pH sensitivity, pH and PIP2 gating kinetics, as well as a K+-dependent inactivation process at the selectivity filter and therefore many of the key regulatory mechanisms of Kir channel physiology.     

Effect of sibutramine HCl on cardiac hERG K+ channel

Common clinically used drugs block the delayed rectifier K(+) channels and prolong the cardiac action potential duration associated with long QT syndrome. Here, we investigated the mechanism of hERG K(+) channel current (I(hERG)) blockade expressed in HEK-293 cells by sibutramine HCl, a serotonin-norepinephrine reuptake inhibitor. Sibutramine HCl inhibited I (hERG) in a concentration-dependent manner with the half-maximal inhibitory concentration (IC(50)) value of 2.5 microM at -40 mV. I(hERG) inhibition by sibutramine HCl showed weak voltage dependency, but the time-dependence of I(hERG) inhibition was developed relatively rapidly on membrane depolarization. On hERG channel gating for the S6 and pore regions, the S6 residue hERG mutant Y652A and F656A largely reduced the blocking potency of I(hERG), unlike the pore-region mutants T623A and S624A. These results indicate that sibutramine HCl preferentially inhibits the hERG potassium channel through the residue Y652 and F656, in a supratherapeutic concentration should be avoided by patients with high susceptibility for cardiac arrhythmia.     

hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656

The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is little mechanistic information on this action. We have investigated in detail hERG K(+) channel current (I(hERG)) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell I(hERG) measurements were made at 37 degrees C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I(hERG) tails at -40mV following a 2s depolarization to +20mV with an IC(50) value of 80nM. Comparable levels of I(hERG) inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I(hERG) was only weakly voltage-dependent, though the time dependence of I(hERG) inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I(hERG) inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K(+) channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore.     

External Ba2+ block of the two-pore domain potassium channel TREK-1 defines conformational transition in its selectivity filter

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.     

Voltage-dependent gating at the KcsA selectivity filter

The prokaryotic K(+) channel KcsA, although lacking a 'standard' voltage-sensing domain, shows voltage-dependent gating that leads to an increase in steady-state open probability of almost two orders of magnitude between +150 and -150 mV. Here we show that voltage-dependent gating in KcsA is associated with the movement of approximately 0.7 equivalent electronic charges. This charge movement produces an increase in the rate of entry into a long-lived inactivated state and seems to be independent of the proton-activation mechanism. Charge neutralization at position 71 renders the channel essentially voltage-independent by preventing entry into the inactivated state. A mechanism for voltage-dependent gating at the selectivity filter is proposed that is based on the reorientation of the carboxylic moiety of Glu71 and its influence in the conformational dynamics of the selectivity filter.     

Saxitoxin is a gating modifier of HERG K+ channels

Potassium (K+) channels mediate numerous electrical events in excitable cells, including cellular membrane potential repolarization. The hERG K+ channel plays an important role in myocardial repolarization, and inhibition of these K+ channels is associated with long QT syndromes that can cause fatal cardiac arrhythmias. In this study, we identify saxitoxin (STX) as a hERG channel modifier and investigate the mechanism using heterologous expression of the recombinant channel in HEK293 cells. In the presence of STX, channels opened slower during strong depolarizations, and they closed much faster upon repolarization, suggesting that toxin-bound channels can still open but are modified, and that STX does not simply block the ion conduction pore. STX decreased hERG K+ currents by stabilizing closed channel states visualized as shifts in the voltage dependence of channel opening to more depolarized membrane potentials. The concentration dependence for steady-state modification as well as the kinetics of onset and recovery indicate that multiple STX molecules bind to the channel. Rapid application of STX revealed an apparent "agonist-like" effect in which K+ currents were transiently increased. The mechanism of this effect was found to be an effect on the channel voltage-inactivation relationship. Because the kinetics of inactivation are rapid relative to activation for this channel, the increase in K+ current appeared quickly and could be subverted by a decrease in K+ currents due to the shift in the voltage-activation relationship at some membrane potentials. The results are consistent with a simple model in which STX binds to the hERG K+ channel at multiple sites and alters the energetics of channel gating by shifting both the voltage-inactivation and voltage-activation processes. The results suggest a novel extracellular mechanism for pharmacological manipulation of this channel through allosteric coupling to channel gating.     

Na+ permeation and block of hERG potassium channels

The inactivation gating of hERG channels is important for the channel function and drug-channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms.     

A gate in the selectivity filter of potassium channels

The selectivity filter of potassium channels is the structural element directly responsible for the selective and rapid conduction of K+, whereas other parts of the protein are thought to function as a molecular gate that either permits or blocks the passage of ions. However, whether the selectivity filter itself also possesses the ability to play the role of a gate is an unresolved question. Using free energy molecular dynamics simulations, it is shown that the reorientation of two peptide linkages in the selectivity filter of the KcsA K+ channel can lead to a stable nonconducting conformational state. Two microscopic factors influence the transition toward such a conformational state: the occupancy of one specific cation binding site in the selectivity filter (S2), and the strength of intersubunit interactions involving the GYG signature sequence. These results suggest that such conformational transitions occurring in the selectivity filter might be related to different K+ channel gating events, including C-type (slow) inactivation.     

Human ether-a-go-go related gene (hERG) K channels: Function and dysfunction

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in regulating cardiac excitability and maintenance of normal cardiac rhythm. Mutations in hERG cause a third of all cases of congenital long QT syndrome, a disorder of cardiac repolarisation characterised by prolongation of the QT interval on the surface electrocardiogram, abnormal T waves, and a risk of sudden cardiac death due to ventricular arrhythmias. Additionally, the hERG channel protein is the molecular target for almost all drugs that cause the acquired form of long QT syndrome. Advances in understanding the structural basis of hERG gating, its traffic to the cell surface, and the molecular architecture involved in drug-block of hERG, are providing the foundation for rational treatment and prevention of hERG associated long QT syndrome. This review summarises the current knowledge of hERG function and dysfunction, and the areas of ongoing research.     

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

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

A molecular switch between the outer and the inner vestibules of the voltage-gated Na+ channel

Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K(+) channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na(+) channels, but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid Ile-1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the adult rat skeletal muscle isoform of the voltage-gated sodium channel (rNa(V)1.4) may act as molecular switch allowing for interaction between outer and inner vestibules. Cysteine scanning mutagenesis of the internal part of DIV-S6 revealed that only mutations at site 1575 rescued the channel from a unique kinetic state ("ultra-slow inactivation," I(US)) produced by the mutation K1237E in the selectivity filter. A similar effect was seen with I1575A. Previously, we reported that conformational changes of both the internal and the external vestibule are involved in the generation of I(US). The fact that mutations at site 1575 modulate I(US) produced by K1237E strongly suggests an interaction between these sites. Our data confirm a previously published molecular model in which Ile-1575 of DIV-S6 is in close proximity to Lys-1237 of the selectivity filter. Furthermore, these functional data define the position of the selectivity filter relative to the adjacent DIV-S6 segment within the ionic permeation pathway.     

Mechanistic insight into human ether-à-go-go-related gene (hERG) K+ channel deactivation gating from the solution structure of the EAG domain

Human ether-à-go-go-related gene (hERG) K(+) channels have a critical role in cardiac repolarization. hERG channels close (deactivate) very slowly, and this is vital for regulating the time course and amplitude of repolarizing current during the cardiac action potential. Accelerated deactivation is one mechanism by which inherited mutations cause long QT syndrome and potentially lethal arrhythmias. hERG deactivation is highly dependent upon an intact EAG domain (the first 135 amino acids of the N terminus). Importantly, deletion of residues 2-26 accelerates deactivation to a similar extent as removing the entire EAG domain. These and other experiments suggest the first 26 residues (NT1-26) contain structural elements required to slow deactivation by stabilizing the open conformation of the pore. Residues 26-135 form a Per-Arnt-Sim domain, but a structure for NT1-26 has not been forthcoming, and little is known about its site of interaction on the channel. In this study, we present an NMR structure for the entire EAG domain, which reveals that NT1-26 is structurally independent from the Per-Arnt-Sim domain and contains a stable amphipathic helix with one face being positively charged. Mutagenesis and electrophysiological studies indicate that neutralizing basic residues and breaking the amphipathic helix dramatically accelerate deactivation. Furthermore, scanning mutagenesis and molecular modeling studies of the cyclic nucleotide binding domain suggest that negatively charged patches on its cytoplasmic surface form an interface with the NT1-26 domain. We propose a model in which NT1-26 obstructs gating motions of the cyclic nucleotide binding domain to allosterically stabilize the open conformation of the pore.     

Proton block of the pore underlies the inhibition of hERG cardiac K+ channels during acidosis

Human ether-a-go-go-related gene (hERG) potassium channels are critical determinants of cardiac repolarization. Loss of function of hERG channels is associated with Long QT Syndrome, arrhythmia, and sudden death. Acidosis occurring as a result of myocardial ischemia inhibits hERG channel function and may cause a predisposition to arrhythmias. Acidic pH inhibits hERG channel maximal conductance and accelerates deactivation, likely by different mechanisms. The mechanism underlying the loss of conductance has not been demonstrated and is the focus of the present study. The data presented demonstrate that, unlike in other voltage-gated potassium (Kv) channels, substitution of individual histidine residues did not abolish the pH dependence of hERG channel conductance. Abolition of inactivation, by the mutation S620T, also did not affect the proton sensitivity of channel conductance. Instead, voltage-dependent channel inhibition (δ = 0.18) indicative of pore block was observed. Consistent with a fast block of the pore, hERG S620T single channel data showed an apparent reduction of the single channel current amplitude at low pH. Furthermore, the effect of protons was relieved by elevating external K(+) or Na(+) and could be modified by charge introduction within the outer pore. Taken together, these data strongly suggest that extracellular protons inhibit hERG maximal conductance by blocking the external channel pore.