Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB.

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

Expanding the genetic and phenotypic relevance of KCNB1 variants in developmental and epileptic encephalopathies: 27 new patients and overview of the literature.

Developmental and epileptic encephalopathies (DEE) refer to a heterogeneous group of devastating neurodevelopmental disorders. Variants in KCNB1 have been recently reported in patients with early-onset DEE. KCNB1 encodes the α subunit of the delayed rectifier voltage-dependent potassium channel Kv 2.1. We review the 37 previously reported patients carrying 29 distinct KCNB1 variants and significantly expand the mutational spectrum describing 18 novel variants from 27 unreported patients. Most variants occur de novo and mainly consist of missense variants located on the voltage sensor and the pore domain of Kv 2.1. We also report the first inherited variant (p.Arg583*). KCNB1-related encephalopathies encompass a wide spectrum of neurodevelopmental disorders with predominant language difficulties and behavioral impairment. Eighty-five percent of patients developed epilepsies with variable syndromes and prognosis. Truncating variants in the C-terminal domain are associated with a less-severe epileptic phenotype. Overall, this report provides an up-to-date review of the mutational and clinical spectrum of KCNB1, strengthening its place as a causal gene in DEEs and emphasizing the need for further functional studies to unravel the underlying mechanisms.

Spectrum of KV 2.1 Dysfunction in KCNB1-Associated Neurodevelopmental Disorders.

OBJECTIVE: Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel KV 2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. METHODS: We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant KV 2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. RESULTS: Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild-type KV 2.1. Quantification of protein expression also identified variants with reduced total KV 2.1 expression or deficient cell-surface expression. INTERPRETATION: Our study establishes a platform for rapid screening of KV 2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype. ANN NEUROL 2019;86:899-912.

Oxidation of KCNB1 potassium channels in the murine brain during aging is associated with cognitive impairment.

Voltage-gated potassium (K+) channel sub-family B member 1 (KCNB1, Kv2.1) is known to undergo oxidation-induced oligomerization during aging but whether this process affects brain's physiology was not known. Here, we used 10, 16 and 22 month-old transgenic mice overexpressing a KCNB1 variant that does not oligomerize (Tg-C73A) and as control, mice overexpressing the wild type (Tg-WT) channel and non-transgenic (non-Tg) mice to elucidate the effects of channel's oxidation on cognitive function. Aging mice in which KCNB1 oligomerization is negligible (Tg-C73A), performed significantly better in the Morris Water Maze (MWM) test of working memory compared to non-Tg or Tg-WT mice. KCNB1 and synapsin-1 co-immunoprecipitated and the cognitive impairment in the MWM was associated with moderate loss of synapsin-1 in pre-synaptic structures of the hippocampus, whereas neurodegeneration and neuronal loss were not significantly different in the various genotypes. We conclude that moderate oxidation of the KCNB1 channel during aging can influence neuronal networks by affecting synaptic function.

The Insensitivity of TASK-3 K2P Channels to External Tetraethylammonium (TEA) Partially Depends on the Cap Structure.

Two-pore domain K⁺ channels (K2P) display a characteristic extracellular cap structure formed by two M1-P1 linkers, the functional role of which is poorly understood. It has been proposed that the presence of the cap explains the insensitivity of K2P channels to several K⁺ channel blockers including tetraethylammonium (TEA). We have explored this hypothesis using mutagenesis and functional analysis, followed by molecular simulations. Our results show that the deletion of the cap structure of TASK-3 (TWIK-related acid-sensitive K⁺ channel) generates a TEA-sensitive channel with an IC50 of 11.8 ± 0.4 mM. The enhanced sensitivity to TEA displayed by the cap-less channel is also explained by the presence of an extra tyrosine residue at position 99. These results were corroborated by molecular simulation analysis, which shows an increased stability in the binding of TEA to the cap-less channel when a ring of four tyrosine is present at the external entrance of the permeation pathway. Consistently, Y99A or Y205A single-residue mutants generated in a cap-less channel backbone resulted in TASK-3 channels with low affinity to external TEA.

Novel KCNB1 mutation associated with non-syndromic intellectual disability.

Potassium voltage-gated channel subfamily B member 1 (KCNB1) encodes Kv2.1 potassium channel of crucial role in hippocampal neuron excitation homeostasis. KCNB1 mutations are known to cause early-onset infantile epilepsy. To date, 10 KCNB1 mutations have been described in 11 patients. Using whole-exome sequencing, we identified a novel de novo missense (c.1132G>C, p.V378L) KCNB1 mutation in a patient with global developmental delay, intellectual disability, severe speech impairment, but no episode of epilepsy until the lastly examined age of 6 years old. Furthermore, she showed neuropsychiatric symptoms including hyperactivity with irritability, heteroaggressiveness, psychomotor instability and agitation. Our observation might expand the phenotypic spectrum of KCNB1-related phenotypes and raises the issue of the occurrence of the epileptic phenotype.

Syntaxin-binding domain of Kv2.1 is essential for the expression of apoptotic K+ currents.

Intracellular signalling cascades triggered by oxidative injury can lead to upregulation of Kv2.1 K(+) channels at the plasma membrane of dying neurons. Membrane incorporation of new channels is necessary for enhanced K(+) efflux and a consequent reduction of intracellular K(+) that facilitates apoptosis. We showed previously that the observed increase in K(+) currents is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated process, and that the SNARE protein syntaxin binds directly to Kv2.1 channels. In the present study, we tested whether disrupting the interaction of Kv2.1 and syntaxin promoted the survival of cortical neurons following injury. Syntaxin is known to bind to Kv2.1 in a domain comprising amino acids 411-522 of the channel's cytoplasmic C terminus (C1a). Here we show that this domain is required for the apoptotic K(+) current enhancement. Moreover, expression of an isolated, Kv2.1-derived C1a peptide is sufficient to suppress the injury-induced increase in currents by interfering with Kv2.1/syntaxin binding. By subdividing the C1a peptide, we were able to localize the syntaxin binding site on Kv2.1 to the most plasma membrane-distal residues of C1a. Importantly, expression of this peptide segment in neurons prevented the apoptotic K(+) current enhancement and cell death following an oxidative insult, without greatly impairing baseline K(+) currents or normal electrical profiles of neurons. These results establish that binding of syntaxin to Kv2.1 is crucial for the manifestation of oxidant-induced apoptosis, and thereby reveal a potential new direction for therapeutic intervention in the treatment of neurodegenerative disorders.

Dynamics of hERG closure allow novel insights into hERG blocking by small molecules.

Today, drug discovery routinely uses experimental assays to determine very early if a lead compound can yield certain types of off-target activity. Among such off targets is hERG. The ion channel plays a primordial role in membrane repolarization and altering its activity can cause severe heart arrhythmia and sudden death. Despite routine tests for hERG activity, rather little information is available for helping medicinal chemists and molecular modelers to rationally circumvent hERG activity. In this article novel insights into the dynamics of hERG channel closure are described. Notably, helical pairwise closure movements have been observed. Implications and relations to hERG inactivation are presented. Based on these dynamics novel insights on hERG blocker placement are presented, compared to literature, and discussed. Last, new evidence for horizontal ligand positioning is shown in light of former studies on hERG blockers.

One-shot design elevates functional expression levels of a voltage-gated potassium channel.

Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web-accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native-state energy. As a stringent test, we apply mPROSS to the Kv1.2-Kv2.1 paddle chimera voltage-gated potassium channel. Four designs, encoding 9-26 mutations relative to the parental channel, were functional and maintained potassium-selective permeation and voltage dependence in Xenopus oocytes with up to 14-fold increase in whole-cell current densities. Additionally, single-channel recordings reveal no significant change in the channel-opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamic channels and receptors may be enhanced through one-shot design calculations.

Analysis of the interaction of tarantula toxin Jingzhaotoxin-III (ß-TRTX-Cj1α) with the voltage sensor of Kv2.1 uncovers the molecular basis for cross-activities on Kv2.1 and Nav1.5 channels.

Animal venoms contain a fascinating array of divergent peptide toxins that have cross-activities on different types of voltage-gated ion channels. However, the underlying mechanism remains poorly understood. Jingzhaotoxin-III (JZTX-III), a 36-residue peptide from the tarantula Chilobrachys jingzhao, is specific for Nav1.5 and Kv2.1 channels over the majority of other ion channel subtypes. JZTX-III traps the Nav1.5 DII voltage sensor at closed state by binding to the DIIS3-S4 linker. In this study, electrophysiological experiments showed that JZTX-III had no effect on five voltage-gated potassium channel subtypes (Kv1.4, Kv3.1, and Kv4.1-4.3), whereas it significantly inhibited Kv2.1 with an IC50 of 0.71 ± 0.01 µM. Mutagenesis and modeling data suggested that JZTX-III docks at the Kv2.1 voltage-sensor paddle. Alanine replacement of Phe274, Lys280, Ser281, Leu283, Gln284, and Val288 could decrease JZTX-III affinity by 7-, 9-, 34-, 12-, 9-, and 7-fold, respectively. Among them, S281 is the most crucial determinant, and the substitution with Thr only slightly reduced toxin sensitivity. In contrast, a single conversion of Ser281 to Ala, Phe, Ile, Val, or Glu increased the IC50 value by >34-fold. Alanine-scanning mutagenesis experiments indicated that the functional surface of JZTX-III bound to the Kv2.1 channel is composed of four hydrophobic residues (Trp8, Trp28, Trp30, and Val33) and three charged residues (Arg13, Lys15, and Glu34). The bioactive surfaces of JZTX-III interacting with Kv2.1 and Nav1.5 are only partially overlapping. These results strongly supported the hypothesis that animal toxins might use partially overlapping bioactive surfaces to target the voltage-sensor paddles of two different types of ion channels. Increasing our understanding of the molecular mechanisms of toxins interacting with voltage-gated sodium and potassium channels may provide new molecular insights into the design of more potent ion channel inhibitors.

Water wires in atomistic models of the Hv1 proton channel.

The voltage-gated proton channel (Hv1) is homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels but lacks a separate pore domain. The Hv1 monomer has dual functions: it gates the proton current and also serves as the proton conduction pathway. To gain insight into the structure and dynamics of the yet unresolved proton permeation pathway, we performed all-atom molecular dynamics simulations of two different Hv1 homology models in a lipid bilayer in excess water. The structure of the Kv1.2-Kv2.1 paddle-chimera VSD was used as template to generate both models, but they differ in the sequence alignment of the S4 segment. In both models, we observe a water wire that extends through the membrane, whereas the corresponding region is dry in simulations of the Kv1.2-Kv2.1 paddle-chimera. We find that the kinetic stability of the water wire is dependent upon the identity and location of the residues lining the permeation pathway, in particular, the S4 arginines. A measurement of water transport kinetics indicates that the water wire is a relatively static feature of the permeation pathway. Taken together, our results suggest that proton conduction in Hv1 may occur via Grotthuss hopping along a robust water wire, with exchange of water molecules between inner and outer ends of the permeation pathway minimized by specific water-protein interactions. This article is part of a Special Issue entitled: Membrane protein structure and function.

Role of outer-pore residue Y380 in U-type inactivation of KV2.1 channels.

The interpretation of slow inactivation in potassium channels has been strongly influenced by work on C-type inactivation in Shaker channels. Slow inactivation in Shaker and some other potassium channels can be dramatically modulated by the state of the pore, including mutations at outer pore residue T449, which altered inactivation kinetics up to 100-fold. KV2.1, another voltage-dependent potassium channel, exhibits a biophysically distinct inactivation mechanism with a U-shaped voltage-dependence and preferential closed-state inactivation, termed U-type inactivation. However, it remains to be demonstrated whether U-type and C-type inactivation have different molecular mechanisms. This study examines mutations at Y380 (homologous to Shaker T449) to investigate whether C-type and U-type inactivation have distinct molecular mechanisms, and whether C-type inactivation can occur at all in KV2.1. Y380 mutants do not introduce C-type inactivation into KV2.1 and have little effect on U-type inactivation of KV2.1. Interestingly, two of the mutants tested exhibit twofold faster recovery from inactivation compared to wild-type channels. The observation that mutations have little effect suggests KV2.1 lacks C-type inactivation as it exists in Shaker and that C-type and U-type inactivation have different molecular mechanisms. Kinetic modeling predicts that all mutants inactivate preferentially, but not exclusively, from partially activated closed states. Therefore, KV2.1 exhibits a single U-type inactivation process including some inactivation from open as well as closed states.

Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment.

Voltage-dependent K+ (Kv) channels repolarize the action potential in neurons and muscle. This type of channel is gated directly by membrane voltage through protein domains known as voltage sensors, which are molecular voltmeters that read the membrane voltage and regulate the pore. Here we describe the structure of a chimaeric voltage-dependent K+ channel, which we call the 'paddle-chimaera channel', in which the voltage-sensor paddle has been transferred from Kv2.1 to Kv1.2. Crystallized in complex with lipids, the complete structure at 2.4 ångström resolution reveals the pore and voltage sensors embedded in a membrane-like arrangement of lipid molecules. The detailed structure, which can be compared directly to a large body of functional data, explains charge stabilization within the membrane and suggests a mechanism for voltage-sensor movements and pore gating.

An in silico investigation of Kv2.1 potassium channel: Model building and inhibitors binding sites analysis.

Kv2.1 is widely expressed in brain, and inhibiting Kv2.1 is a potential strategy to prevent cell death and achieve neuroprotection in ischemic stroke. Herein, an in silico model of Kv2.1 tetramer structure was constructed by employing the AlphaFold-Multimer deep learning method to facilitate the rational discovery of Kv2.1 inhibitors. GaMD was utilized to create an ion transporting trajectory, which was analyzed with HMM to generate multiple representative receptor conformations. The binding site of RY785 and RY796(S) under the P-loop was defined with Fpocket program together with the competitive binding electrophysiology assay. The docking poses of the two inhibitors were predicted with the aid of the semi-empirical quantum mechanical calculation, and the IGMH results suggested that Met375, Thr376, and Thr377 of the P-helix and Ile405 of the S6 segment made significant contributions to the binding affinity. These results provided insights for rational molecular design to develop novel Kv2.1 inhibitors.

Size of cell-surface Kv2.1 domains is governed by growth fluctuations.

The Kv2.1 voltage-gated potassium channel forms stable clusters on the surface of different mammalian cells. Even though these cell-surface structures have been observed for almost a decade, little is known about the mechanism by which cells maintain them. We measure the distribution of domain sizes to study the kinetics of their growth. Using a Fokker-Planck formalism, we find no evidence for a feedback mechanism present to maintain specific domain radii. Instead, the size of Kv2.1 clusters is consistent with a model where domain size is established by fluctuations in the trafficking machinery. These results are further validated using likelihood and Akaike weights to select the best model for the kinetics of domain growth consistent with our experimental data.

KV2.1 and electrically silent KV channel subunits control excitability and contractility of guinea pig detrusor smooth muscle.

Voltage-gated K(+) (K(V)) channels are implicated in detrusor smooth muscle (DSM) function. However, little is known about the functional role of the heterotetrameric K(V) channels in DSM. In this report, we provide molecular, electrophysiological, and functional evidence for the presence of K(V)2.1 and electrically silent K(V) channel subunits in guinea pig DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of the homotetrameric K(V)2.1, K(V)2.2, and K(V)4.2 as well as the heterotetrameric K(V)2.1/6.3 and K(V)2.1/9.3 channels, was used to examine the role of these K(V) channels in DSM function. RT-PCR indicated mRNA expression of K(V)2.1, K(V)6.2-6.3, K(V)8.2, and K(V)9.1-9.3 subunits in isolated DSM cells. K(V)2.1 protein expression was confirmed by Western blot and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the K(V) current in freshly isolated DSM cells. ScTx1 (100 nM) did not significantly change the steady-state activation and inactivation curves for K(V) current. However, ScTx1 (100 nM) decreased the activation time-constant of the K(V) current at positive voltages. Although our patch-clamp data could not exclude the presence of the homotetrameric K(V)2.1 channels, the biophysical characteristics of the ScTx1-sensitive current were consistent with the presence of heterotetrameric K(V)2.1/silent K(V) channels. Current-clamp recordings showed that ScTx1 (100 nM) did not change the DSM cell resting membrane potential. ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and muscle tone as well as the amplitude of the electrical field stimulation-induced contractions of isolated DSM strips. Collectively, our data revealed that K(V)2.1-containing channels are important physiological regulators of guinea pig DSM excitability and contractility.

Dynamic modulation of the kv2.1 channel by SRC-dependent tyrosine phosphorylation.

The voltage-gated K(+) channel Kv2.1 is expressed as a highly phosphorylated protein in most central neurons, where it plays a key role in regulating neuronal membrane excitability. Previous studies have shown that Kv2.1 channel activity is upregulated by Src-mediated phosphorylation through an unknown mechanism. However, a systematic analysis of the molecular mechanism of Kv2.1 channel phosphorylation by Src is lacking. Here, we show that tyrosine phosphorylation by Src plays a fundamental role in regulating Kv2.1-mediated K(+) current enhancement. We found that the level of expression of the Kv2.1 protein is increased by Src kinase. Using mass spectrometric proteomic techniques, we identified two novel phosphotyrosine sites, Y686 and Y810, in the cytoplasmic domains of Kv2.1. We found that Src-dependent phosphorylation at these sites affects Kv2.1 through distinct regulatory mechanisms. Whereas phosphorylation at Y686 regulates Kv2.1 activity similarly to the known site Y124, phosphorylation at Y810 plays a significant role in regulating the intracellular trafficking of Kv2.1 channels. Our results show that these two novel tyrosine phosphorylation sites of Kv2.1 are crucial to regulating diverse aspects of Kv2.1 channel function and provide novel insights into molecular mechanisms for the regulation of Src-dependent modulation of Kv2.1 channels.

Molecular determinants of U-type inactivation in Kv2.1 channels.

Kv2.1 channels exhibit a U-shaped voltage-dependence of inactivation that is thought to represent preferential inactivation from preopen closed states. However, the molecular mechanisms underlying so-called U-type inactivation are unknown. We have performed a cysteine scan of the S3-S4 and S5-P-loop linkers and found sites that are important for U-type inactivation. In the S5-P-loop linker, U-type inactivation was preserved in all mutant channels except E352C. This mutation, but not E352Q, abolished closed-state inactivation while preserving open-state inactivation, resulting in a loss of the U-shaped voltage profile. The reducing agent DTT, as well as the C232V mutation in S2, restored U-type inactivation to the E352C mutant, which suggests that residues 352C and C232 may interact to prevent U-type inactivation. The R289C mutation, in the S3-S4 linker, also reduced U-type inactivation. In this case, DTT had little effect but application of MTSET restored wild-type-like U-type inactivation behavior, suggestive of the importance of charge at this site. Kinetic modeling suggests that the E352C and R289C inactivation phenotypes largely resulted from reductions in the rate constants for transitions from closed to inactivated states. The data indicate that specific residues within the S3-S4 and S5-P-loop linkers may play important roles in Kv2.1 U-type inactivation.

Formation of heteromeric Kv2 channels in mammalian brain neurons.

The formation of heteromeric tetramers is a common feature of voltage-gated potassium (Kv) channels. This results in the generation of a variety of tetrameric Kv channels that exhibit distinct biophysical and biochemical characteristics. Kv2 delayed rectifier channels are, however, unique exceptions. It has been previously shown that mammalian Kv2.1 and Kv2.2 are localized in distinct domains of neuronal membranes and are not capable of forming heteromeric channels with each other (Hwang, P. M., Glatt, C. E., Bredt, D. S., Yellen, G., and Snyder, S. H. (1992) Neuron 8, 473-481). In this study, we report a novel form of rat Kv2.2, Kv2.2(long), which has not been previously recognized. Our data indicate that Kv2.2(long) is the predominant form of Kv2.2 expressed in cortical pyramidal neurons. In contrast to the previous findings, we also found that rat Kv2.1 and Kv2.2(long) are colocalized in the somata and proximal dendrites of cortical pyramidal neurons and are capable of forming functional heteromeric delayed rectifier channels. Our results suggest that the delayed rectifier currents, which regulate action potential firing, are encoded by heteromeric Kv2 channels in cortical neurons.

Overlapping binding sites of structurally different antiarrhythmics flecainide and propafenone in the subunit interface of potassium channel Kv2.1.

Kv2.1 channels, which are expressed in brain, heart, pancreas, and other organs and tissues, are important targets for drug design. Flecainide and propafenone are known to block Kv2.1 channels more potently than other Kv channels. Here, we sought to explore structural determinants of this selectivity. We demonstrated that flecainide reduced the K(+) currents through Kv2.1 channels expressed in Xenopus laevis oocytes in a voltage- and time-dependent manner. By systematically exchanging various segments of Kv2.1 with those from Kv1.2, we determined flecainide-sensing residues in the P-helix and inner helix S6. These residues are not exposed to the inner pore, a conventional binding region of open channel blockers. The flecainide-sensing residues also contribute to propafenone binding, suggesting overlapping receptors for the drugs. Indeed, propafenone and flecainide compete for binding in Kv2.1. We further used Monte Carlo-energy minimizations to map the receptors of the drugs. Flecainide docking in the Kv1.2-based homology model of Kv2.1 predicts the ligand ammonium group in the central cavity and the benzamide moiety in a niche between S6 and the P-helix. Propafenone also binds in the niche. Its carbonyl group accepts an H-bond from the P-helix, the amino group donates an H-bond to the P-loop turn, whereas the propyl group protrudes in the pore and blocks the access to the selectivity filter. Thus, besides the binding region in the central cavity, certain K(+) channel ligands can expand in the subunit interface whose residues are less conserved between K(+) channels and hence may be targets for design of highly desirable subtype-specific K(+) channel drugs.