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.