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.