Molecular mapping of a site for Cd²⁺‐induced modification of human ether‐à‐go‐go‐related gene (hERG) channel activation

Abstract

Cd2+ slows the rate of activation, accelerates the rate of deactivation and shifts the half-points of voltage-dependent activation (V 0.5,act ) and inactivation (V 0.5,inact ) of human ether-à-go-go-related gene (hERG) K + channels. To identify specific Cd 2+ -binding sites on the hERG channel, we mutated potential Cd 2+ -coordination residues located in the transmembrane domains or extracellular loops linking these domains, including five Cys, three His, nine Asp and eight Glu residues. Each residue was individually substituted with Ala and the resulting mutant channels heterologously expressed in Xenopus oocytes and their biophysical properties determined with standard two-microelectrode voltage-clamp technique. Cd 2+ at 0.5 mM caused a +36 mV shift of V 0.5,act and a +18 mV shift of V 0.5,inact in wild-type channels. Most mutant channels had a similar sensitivity to 0.5 mM Cd Human ether-à-go-go-related gene (hERG) channel is a member of the ether-à-go-go (EAG) family of voltage-gated K + channels (Warmke & Ganetzky, 1994) and is expressed in many cell types, including cardiac myocytes, neurones and tumour cells. In cardiomyocytes, hERG channel subunits co-assemble to form homotetrameric channels that conduct the rapid delayed rectifier K + current I Kr (Sanguinetti et al. 1995; Trudeau et al. 1995). I Kr is an important component of the outward currents that mediate repolarization of the cardiac action potential. Inherited loss-of-function mutations in HERG or block of hERG channels by specific drugs prolongs the duration of action potentials and causes long QT syndrome, a disorder of ventricular repolarization that increases the risk of life-threatening arrhythmias (Keating & Sanguinetti, 2001).Divalent cations can modify ion channel gating by screening non-specific negative surface charges located on membrane lipids and proteins (McLaughlin, 1989) or by binding to specific residues of channel proteins (Gilly & Armstrong, 1982). Recent studies have attempted to localize the binding sites using site-directed mutagenesis. Several acidic residues in the S5-P linker of Kv1-Kv4 channels were proposed to account for the majority of negative surface charges that were screened by divalent cations (Elinder et al. 1996(Elinder et al. , 1998. In Drosophila EAG (dEAG) channels, Mg 2+ , Mn 2+ and Ni 2+ slow gating by binding to an extracellular site formed by two acidic residues: D278 in S2 and D327 in S3 (Silverman et al. 2000). Charge pair interactions between these Asp residues and specific basic residues in S4 have been proposed to facilitate activation of dEAG channels and protein folding and activation of Shaker channels (Papazian et al. 1995;Seoh et al. 1996; Tiwari-Woodruff et al. 1997Silverman et al. 2000Silverman et al. , 2003Silverman et al. , 2004. Specific charge pairings may be disrupted and replaced by others as the S4 helix slides past the relatively immobile S2 and S3 domains. In this scheme, divalent cations bind to the Asp residues in S2 or S3 and prevent interaction with S4, causing a slowing of EAG...

Cooperative Interactions Between R531 and Acidic Residues in the Voltage Sensing Module of hERG1 Channels

“…These specific interactions were supported by the recently solved crystal structure of Kv1.2, demonstrating a salt bridge between R303 and E226 (corresponding to R531 and D456 in hERG1, respectively) in the open conformation [21]. Moreover, D456, D460 and D509 in hERG1 form a coordination site for external Cd 2+ implying that these residues are located close to one another [42]. A homology model of hERG1 based on the Kv1.2 crystal structure illustrates the proximity of R531 to this negatively charged pocket in the open state (Fig.…”

Ether-à-go-go family voltage-gated K+ channels evolved in an ancestral metazoan and functionally diversified in a cnidarian–bilaterian ancestor

“…Mutagenic neutralization of these charges in mammalian Eag, Elk and Erg channels depolarizes the voltage-activation curve (Fernandez et al, 2005;Silverman et al, 2000;Zhang et al, 2009). Furthermore, voltage activation of EAG channels is universally depolarized by extracellular protons through neutralization of the EAG acidic charges (Kazmierczak et al, 2013).…”

“…We examined the pH and divalent cation sensitivity of NvErg1 activation because voltage activation of human Erg1 is also inhibited by binding of protons and Ca 2+ and Mg 2+ to the EAG-specific voltage sensor site (Fernandez et al, 2005;Jo et al, 1999;Kazmierczak et al, 2013). The pH sensitivity of human Erg is extremely low at physiological concentrations of Ca 2+ , but increases dramatically if Ca 2+ is reduced to micromolar levels (Kazmierczak et al, 2013).…”

“…Mg 2+ sensitivity of Eag channels is primarily conferred by acidic residues in the voltage sensor (Silverman et al, 2000) that are unique to the EAG family of K + channels, but are shared with hyperpolarizationgated cation channels (HCNs) and cyclic-nucleotide-gated cation channels (CNGs). Erg channel kinetics are sensitive to both Mg 2+ and Ca 2+ (Fernandez et al, 2005;Jo et al, 1999), whereas Elk channels are insensitive to these ions, but are inhibited by Zn 2+ (Zhang et al, 2009). Protons inhibit voltage-dependent activation of all EAG family channels examined so far through protonation of these same EAG family acidic residues (Kazmierczak et al, 2013) and compete with divalent cations for the binding site (Kazmierczak et al, 2013;Terlau et al, 1996).…”

Molecular Determinants of Pentamidine-Induced hERG Trafficking Inhibition

“…Guided by our rescue experiments, we surveyed a series of mutations in the transmembrane domain of hERG (S1-S6) that replaced negatively charged glutamate or aspartate residues with alanine, because we considered negatively charged amino acid residues putative interaction partners for positively charged pentamidine. Mutations in the S1-S4 voltage sensor domain included hERG 4EC, which removes not one but four glutamate residues in the extracellular S1-S2 linker (Fernandez et al, 2005), hERG D456A, D460A, D466A, D509A, E518A, E519A, D540A, and E544A. In the inner pore region (S5-S6), we tested three additional negative charge replacements, hERG E575A, D580A, and D591A.…”