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 Ba(2+) (Ba(2+) (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 Ba(2+) (Ba(2+) (o)) blocks K(+) channel pores, and thereby serves as a useful probe of K(+) channel structure. HERG channel inactivation promotes relief of Ba(2+) 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 Ba(2+) (o) block by promoting inactivation. Instead, Na(+) (o) slowed Ba(2+) egress and Ba(2+) 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 Ba(2+) 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.