Sodium channels are principal molecular determinants responsible for myocardial conduction and maintenance of the cardiac rhythm. Calcium ions (Ca2+) have a fundamental role in the coupling of cardiac myocyte excitation and contraction, yet mechanisms whereby intracellular Ca2+ may directly modulate Na channel function have yet to be identified. Here we show that calmodulin (CaM), a ubiquitous Ca2+-sensing protein, binds to the carboxy-terminal 'IQ' domain of the human cardiac Na channel (hH1) in a Ca2+-dependent manner. This binding interaction significantly enhances slow inactivation-a channel-gating process linked to life-threatening idiopathic ventricular arrhythmias. Mutations targeted to the IQ domain disrupted CaM binding and eliminated Ca2+/CaM-dependent slow inactivation, whereas the gating effects of Ca2+/CaM were restored by intracellular application of a peptide modelled after the IQ domain. A naturally occurring mutation (A1924T) in the IQ domain altered hH1 function in a manner characteristic of the Brugada arrhythmia syndrome, but at the same time inhibited slow inactivation induced by Ca2+/CaM, yielding a clinically benign (arrhythmia free) phenotype.

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Mutations that disrupt Na+ channel fast inactivation attenuate lidocaine (lignocaine)‐induced use dependence; however, the pharmacological role of slower inactivation processes remains unclear. In Xenopus oocytes, tryptophan substitution in the outer pore of the rat skeletal muscle channel (μ1‐W402) alters partitioning among fast‐ and slow‐inactivated states. We therefore examined the effects of W402 mutations on lidocaine block. Recovery from inactivation exhibited three kinetic components (IF, fast; IM, intermediate; IS, slow). The effects of W402A and W402S on IF and IS differed, but both mutants (with or without β1 subunit coexpression) decreased the amplitude of IM. In wild‐type channels, lidocaine imposed a delayed recovery component with intermediate kinetics, and use‐dependent block was attenuated in both W402A and W402S. To examine the pharmacological role of IS relative to IM, drug‐exposed β1‐coexpressed channels were subjected to 2 min depolarizations. Lidocaine had no effect on sodium current (INa) after a 1 s hyperpolarization interval that allowed recovery from IM but not IS, suggesting that lidocaine affinity for IS is low. Both W402 mutations reduced occupancy of IM in drug‐free conditions, and also induced resistance to use‐dependent block. We propose that lidocaine‐induced use dependence may involve an allosteric conformational change in the outer pore.

Most voltage-gated K+ currents are relatively insensitive to extracellular Na+ (Na+ o), but Na+ o potently inhibits outward human ether-a-go-go–related gene (HERG)–encoded K+ channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429–30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na+ (Na+ i) and extracellular Ba2+ (Ba2+ o), to define a site where Na+ o interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na+ o was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K+ selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na+ o. Quantitatively similar effects of Na+ i to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na+ o sensitivity, this result suggested that Na+ o and Na+ i act at different sites. Extracellular Ba2+ (Ba2+ o) blocks K+ channel pores, and thereby serves as a useful probe of K+ channel structure. HERG channel inactivation promotes relief of Ba2+ block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265–278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na+ o action. A remote allosteric model predicts that Na+ o will speed relief of Ba2+ o block by promoting inactivation. Instead, Na+ o slowed Ba2+ egress and Ba2+ relieved Na+ o inhibition, consistent with Na+ o binding to an outer pore site. The apparent affinities of the outer pore for Na+ o and K+ o as measured by slowing of Ba2+ egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na+ o inhibition. Na+ o inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.

Human ether-a-go-go-related gene (HERG) encodes the pore-forming subunit of IKr, a cardiac K ؉ channel. Although many commonly used drugs block IKr, in certain individuals, this action evokes a paradoxical life-threatening cardiac rhythm disturbance, known as the acquired long QT syndrome (aLQTS). Although aLQTS has become the leading cause of drug withdrawal by the U.S. Food and Drug Administration, DNA sequencing in aLQTS patients has revealed HERG mutations only in rare cases, suggesting that unknown HERG modulators are often responsible. By using the worm Caenorhabditis elegans, we have developed in vivo behavioral assays that identify candidate modulators of unc-103, the worm HERG orthologue. By using RNA-interference methods, we have shown that worm homologues of two HERG-interacting proteins, Hyperkinetic and K channel regulator 1 (KCR1), modify unc-103 function. Examination of the human KCR1 sequence in patients with drug-induced cardiac repolarization defects revealed a sequence variation (the substitution of isoleucine 447 by valine, I447V) that occurs at a reduced frequency (1.1%) relative to a matched control population (7.0%), suggesting that I447V may be an allele for reduced aLQTS susceptibility. This clinical result is supported by in vitro studies of HERG dofetilide sensitivity by using coexpression of HERG with wild-type and I447V KCR1 cDNAs. Our studies demonstrate the feasibility of using C. elegans to assay and potentially identify aLQTS candidate genes.C aenorhabditis elegans unc-103 shares 70% amino acid identity with HERG in the conserved transmembrane and pore regions of the protein (see Scheme 1).Studies using unc-103 promoter GFP reporter constructs reveal expression of unc-103 in body-wall muscle, egg-laying muscles, pharyngeal muscles, and neurons that innervate these tissues (D.J.R, J.H.T., and R. Garcia, unpublished data). Worm strains carrying mutations in this gene, unc-103 (n500) and unc-103 (e1597), were isolated in screens for locomotion-defective mutants (1). Analysis of both of these neomorphic mutant strains revealed the same mutation: conversion of a conserved alanine in the S6 transmembrane domain to a threonine (A334T, indicated in bold above). Our studies reveal that these mutant worms exhibit profound neuromuscular defects, and the severity of these defects is sensitive to modulators that decrease the level of mutant channel activity. Further, the human homologues of these modulators may have physiologically relevant interactions with human ether-a-go-go-related gene (HERG) and represent acquired long QT syndrome (aLQTS) candidate genes. MethodsMolecular Biology. The human K channel regulator 1 (KCR1) cDNA, an IMAGE clone (no. 650823) was purchased from Research Genetics (Huntsville, AL). Site-directed mutagenesis was performed as described (2). For in vitro cRNA transcription, we used the SP6 mMessage mMachine high-yield capped RNA transcription kit (Ambion, Austin, TX). For RNA interference (RNAi) vectors, we PCR-amplified fragments of 0.9-1.5 kb of the target gene f...