Proton Block of the Pore Underlies the Loss of hERG Channel Conductance during Acidosis

Abstract

Acidic pH reduces conductance in a number of voltage-gated potassium (Kv) channels via stabilization of inactivated states. However, the site and mechanism of action of protons responsible for inhibition of human ether-a-go-go related gene (hERG) Kv channels is unclear. Consistent with previous observations that suggest that a stabilization of inactivated states is not responsible, we show that protons reduce maximal conductance in S620T inactivation-removed channels to the same extent as WT hERG channels. A Woodhull characterization of the voltage-dependence of channel inhibition indicate that protons experience ∼20% of the electric field (δ=0.18) at their binding site. An apparent reduction of the single channel conductance was observed in measurements of hERG single channel activity. These data suggest that rapid block of the hERG channel pore underlies the proton-mediated loss of conductance. Consistent with a block of the outer pore, external K+ (Kd=1 mM, pH5.5) or Na+ relieved the effect of acidic pH. The negatively charged residue E637 in the outer pore mouth is a potential proton binding site; however, we could not test this since neutralization of this site resulted in non-functional channels. As an alternative strategy, we engineered a glutamate at H587, a site previously shown to face into the outer mouth of the pore. H587E reduced the voltage dependence of the proton block indicating that the binding site was shifted to a site that experiences little of the electric field. This implies that the proton binding site in WT channels lies deeper within the pore than H587. Taken together, these data suggest that extracellular protons inhibit hERG maximal conductance by blocking the external channel pore.