Svetlana Z. Stepanovic

A calcium sensor in the sodium channel modulates cardiac excitability

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.

Mechanistic link between lidocaine block and inactivation probed by outer pore mutations in the rat μ1 skeletal muscle sodium channel

1 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. 2 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. 3 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. 4 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.

Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin

Sodium channels are fundamental signaling molecules in excitable cells, and are molecular targets for local anesthetic agents and intracellular free Ca2+ ([Ca2+]i). Two regions of NaV1.5 have been identified previously as [Ca2+]i-sensitive modulators of channel inactivation. These include a C-terminal IQ motif that binds calmodulin (CaM) in different modes depending on Ca2+ levels, and an immediately adjacent C-terminal EF-hand domain that directly binds Ca2+. Here we show that a mutation of the IQ domain (A1924T; Brugada Syndrome) that reduces CaM binding stabilizes NaV1.5 inactivation, similarly and more extensively than even reducing [Ca2+]i. Because the DIII-DIV linker is an essential structure in NaV1.5 inactivation, we evaluated this domain for a potential CaM binding interaction. We identified a novel CaM binding site within the linker, validated its interaction with CaM by NMR spectroscopy, and revealed its micromolar affinity by isothermal titration calorimetry. Mutation of three consecutive hydrophobic residues (Phe1520-Ile1521-Phe1522) to alanines in this CaM-binding domain recapitulated the electrophysiology phenotype observed with mutation of the C-terminal IQ domain: NaV1.5 inactivation was stabilized; moreover, mutations of either CaM-binding domain abolish the well described stabilization of inactivation by lidocaine. The direct physical interaction of CaM with the C-terminal IQ domain and the DIII-DIV linker, combined with the similarity in phenotypes when CaM-binding sites in either domain are mutated, suggests these cytoplasmic structures could be functionally coupled through the action of CaM. These findings have bearing upon Na+ channel function in genetically altered channels and under pathophysiologic conditions where [Ca2+]i impacts cardiac conduction.

Extracellular sodium interacts with the HERG channel at an outer pore site.

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.

Identification and characterization of a compound that protects cardiac tissue from human Ether-à-go-go-related gene (hERG)-related drug-induced arrhythmias

Background: Inhibition of the cardiac hERG channel by essential pharmaceuticals is unpredictable and leads to fatal arrhythmias. Results: Pretreatment with a newly identified compound, VU0405601, reduces sensitivity of hERG to inhibition by multiple blockers and prevents arrhythmias. Conclusion: hERG-related arrhythmias are amenable to preventive therapy. Significance: A novel approach at ion channel modulation that impacts drug discovery and safety concerns is outlined. The human Ether-à-go-go-related gene (hERG)-encoded K+ current, IKr is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes IKr block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC70 of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC50 of dofetilide from 38.7 to 76.3 nm. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

The evolutionarily conserved residue A653 plays a key role in HERG channel closing

Human ether‐a‐go‐go‐related gene (HERG) encodes the rapid, outwardly rectifying K+ current IKr that is critical for repolarization of the cardiac action potential. Congenital HERG mutations or unintended pharmaceutical block of IKr can lead to life‐threatening arrhythmias. Here, we assess the functional role of the alanine at position 653 (HERG‐A653) that is highly conserved among evolutionarily divergent K+ channels. HERG‐A653 is close to the ‘glycine hinge’ implicated in K+ channel opening, and is flanked by tyrosine 652 and phenylalanine 656, which contribute to the drug binding site. We substituted an array of seven (I, C, S, G, Y, V and T) amino acids at position 653 and expressed individual variants in heterologous systems to assess changes in gating and drug binding. Substitution of A653 resulted in negative shifts of the V1/2 of activation ranging from −23.6 (A653S) to −62.5 (A653V) compared to −11.2 mV for wild‐type (WT). Deactivation was also drastically altered: channels with A653I/C substitutions exhibited delayed deactivation in response to test potentials above the activation threshold, while A653S/G/Y/V/T failed to deactivate under those conditions and required hyperpolarization and prolonged holding potentials at −130 mV. While A653S/G/T/Y variants showed decreased sensitivity to the IKr inhibitor dofetilide, these changes could not be correlated with defects in channel closure. Homology modelling suggests that in the closed state, A653 forms tight contacts with several residues from the neighbouring subunit in the tetramer, playing a key role in S6 helix packing at the narrowest part of the vestibule. Our study suggests that A653 plays an important functional role in the outwardly rectifying gating behaviour of HERG, supporting channel closure at membrane potentials negative to the channel activation threshold.

Genetic Screening in C. Elegans Identifies Rho-GTPAse Activating Protein 6 as Novel HERG Regulator

The human ether-a-go-go related gene (HERG) constitutes the pore forming subunit of I(Kr), a K(+) current involved in repolarization of the cardiac action potential. While mutations in HERG predispose patients to cardiac arrhythmias (Long QT syndrome; LQTS), altered function of HERG regulators are undoubtedly LQTS risk factors. We have combined RNA interference with behavioral screening in Caenorhabditis elegans to detect genes that influence function of the HERG homolog, UNC-103. One such gene encodes the worm ortholog of the rho-GTPase activating protein 6 (ARHGAP6). In addition to its GAP function, ARHGAP6 induces cytoskeletal rearrangements and activates phospholipase C (PLC). Here we show that I(Kr) recorded in cells co-expressing HERG and ARHGAP6 was decreased by 43% compared to HERG alone. Biochemical measurements of cell-surface associated HERG revealed that ARHGAP6 reduced membrane expression of HERG by 35%, which correlates well with the reduction in current. In an atrial myocyte cell line, suppression of endogenous ARHGAP6 by virally transduced shRNA led to a 53% enhancement of I(Kr). ARHGAP6 effects were maintained when we introduced a dominant negative rho-GTPase, or ARHGAP6 devoid of rhoGAP function, indicating ARHGAP6 regulation of HERG is independent of rho activation. However, ARHGAP6 lost effectiveness when PLC was inhibited. We further determined that ARHGAP6 effects are mediated by a consensus SH3 binding domain within the C-terminus of HERG, although stable ARHGAP6-HERG complexes were not observed. These data link a rhoGAP-activated PLC pathway to HERG membrane expression and implicate this family of proteins as candidate genes in disorders involving HERG.

Functional interaction between extracellular sodium, potassium and inactivation gating in HERG channels

We have studied the interaction between extracellular K+ (K+o) and extracellular Na+ (Na+o) in human ether‐à‐go‐go related gene (HERG)‐encoded K+ channels expressed in Chinese hamster ovary (CHO‐K1) cells, using the whole‐cell voltage clamp technique. Prior studies indicate that Na+o potently inhibits HERG current (IC50 3 mm) by binding to an outer pore site, and also speeds recovery from inactivation. In this study, we sought to explore the relationship between the Na+o effect on recovery and Na+o inhibition of HERG current, and to determine whether inactivation gating plays a critical role in Na+o inhibition of HERG current. Na+o concentration–response relationships for current inhibition and speeding of recovery were different, with Na+o less potent at speeding recovery. Na+o inhibition of HERG current was relieved by physiological [K+]o, while Na+o speeded recovery from inactivation similarly in the absence or presence of physiological [K+]o. To examine the link between Na+o block and inactivation using an independent approach, we studied hyperpolarization‐activated currents uncoupled from inactivation in the S4–S5 linker mutant D540K. Depolarization‐activated D540K currents were inhibited by Na+o, while hyperpolarization‐activated currents were augmented by Na+o. This result reveals a direct link between Na+o inhibition and a depolarization‐induced conformational change, most likely inactivation. We attempted to simulate the disparate concentration–response relationships for the two effects of Na+o using a kinetic model that included Na+o site(s) affected by permeation and gating. While a model with only a single dynamic Na+o site was inadequate, a model with two distinct Na+o sites was sufficient to reproduce the data.

Characterization of a Compound that Reduces Sensitivity to HERG Inhibitors and Prevents hERG-Related, Drug-Induced Arrhythmias in Isolated Rabbit Hearts

The hERG-encoded K+ current, IKr is essential for repolarization of the cardiac action potential but also a source of cardiotoxicity. Unintended HERG inhibition by diverse pharmaceutical agents can cause the acquired Long QT Syndrome (aLQTS) with ventricular arrhythmias and sudden cardiac death. As such, hERG inhibition represents one of the major reasons for adverse drug events and drug withdrawal from the U.S. market. Indeed, the FDA requires testing of all new drugs for hERG-related pro-arrhythmic potential. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC70 of dofetilide, a well-characterized hERG blocker (PubChem AID1511). One compound (VU0405601), with the desired activity was further characterized using whole-cell patch clamp in stably hERG-expressing HEK cells and found to be a hERG agonist on its own with an EC50 around 11.6 μM. In isolated, Langendorff-perfused rabbit hearts, dofetilide-induced arrhythmias, as well as the spatial dispersion of action potential duration and alternans were reduced following pre-treatment with 50 μM VU0405601. In heterologous hERG-HEK cells, dofetilide block of IKr develops gradually, with continuous pulsing. VU0405601 protects hERG from dofetilide inhibition by increasing the IC50 of dofetilide from 38.7 nM in the absence to 76.3 nM in the presence of VU0405601. Investigation of structure activity relationships surrounding VU0405601 revealed three key structural components: a 3-pyridyl amine moiety, an α-amidoether and a 2-substituted naphthyridine structure (substituted with either halogen or methyl) that are important for protection from dofetilide inhibition. Development of a small molecule that could be co-administered to decrease the risk of arrhythmias in response to HERG inhibitors would improve public health and greatly facilitate the drug discovery process.