External pH modulates EAG superfamily K+ channels through EAG-specific acidic residues in the voltage sensor

Abstract

The Ether-a-go-go (EAG) superfamily of voltage-gated K+ channels consists of three functionally distinct gene families (Eag, Elk, and Erg) encoding a diverse set of low-threshold K+ currents that regulate excitability in neurons and muscle. Previous studies indicate that external acidification inhibits activation of three EAG superfamily K+ channels, Kv10.1 (Eag1), Kv11.1 (Erg1), and Kv12.1 (Elk1). We show here that Kv10.2, Kv12.2, and Kv12.3 are similarly inhibited by external protons, suggesting that high sensitivity to physiological pH changes is a general property of EAG superfamily channels. External acidification depolarizes the conductance–voltage (GV) curves of these channels, reducing low threshold activation. We explored the mechanism of this high pH sensitivity in Kv12.1, Kv10.2, and Kv11.1. We first examined the role of acidic voltage sensor residues that mediate divalent cation block of voltage activation in EAG superfamily channels because protons reduce the sensitivity of Kv12.1 to Zn2+. Low pH similarly reduces Mg2+ sensitivity of Kv10.1, and we found that the pH sensitivity of Kv11.1 was greatly attenuated at 1 mM Ca2+. Individual neutralizations of a pair of EAG-specific acidic residues that have previously been implicated in divalent block of diverse EAG superfamily channels greatly reduced the pH response in Kv12.1, Kv10.2, and Kv11.1. Our results therefore suggest a common mechanism for pH-sensitive voltage activation in EAG superfamily channels. The EAG-specific acidic residues may form the proton-binding site or alternatively are required to hold the voltage sensor in a pH-sensitive conformation. The high pH sensitivity of EAG superfamily channels suggests that they could contribute to pH-sensitive K+ currents observed in vivo.

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

“…5H). NvEag retains high pHsensitivity of voltage activation, which is characteristic of EAG family channels, despite the loss of the S3 EAG acidic residues that contributes to the relevant proton binding site (Kazmierczak et al, 2013). However, the shift in V 50 from pH 8 to pH 6 at 0 mmol l (Fig.…”

“…We quantified the effect of Mg Table 1. Because protons compete with divalent cations at the acidic residue binding site and also inhibit the voltage activation of EAG superfamily channels (Kazmierczak et al, 2013;Terlau et al, 1996), we examined the shift in V 50 caused by 5 mmol l −1 Mg 2+ at pH 8 to maximize Mg 2+ sensitivity, and compared it with the V 50 shift caused by 5 mmol l…”

“…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).…”

“…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). NvErg1 has a charge preserving Asp to Glu substitution at the EAG S3 acidic site (Fig.…”

“…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). Only vertebrate Elk channels have been expressed to date; they share a hyperpolarized activation threshold but variable inactivation, making the ancestral gating phenotype less clear (Engeland et al, 1998;Trudeau et al, 1999;Zou et al, 2003).…”