We have studied the interaction between extracellular K+ (K+o) and extracellular Na+ (Na+o) in human ether-a-go-go related gene (HERG) encoded K+ channels expressed in Chinese hamster ovary (CHO-K1) cells, using the whole-cell voltage clamp technique. Prior studies indicate that Na+o potently inhibits HERG current (IC50 ~ 3 mM) by binding to an outer pore site, and also speeds recovery from inactivation. In this study, we sought to explore the relationship between the Na+o effect on recovery and Na+o inhibition of HERG current, and to determine whether inactivation gating plays a critical role in Na+o inhibition of HERG current. Na+o concentration-response relationships for current inhibition and speeding of recovery were different, with Na+o less potent at speeding recovery. Na+o inhibition of HERG current was relieved by physiologic [K+]o, while Na+o speeded recovery from inactivation similarly in the absence or presence of physiologic [K+]o. To examine the link between Na+o block and inactivation using an independent approach, we studied hyperpolarization-activated currents uncoupled from inactivation in the S4-S5 linker mutant D540K. Depolarization-activated D540K currents were inhibited by Na+o, while hyperpolarization-activated currents were augmented by Na+o. This result reveals a direct link between Na+o inhibition and a depolarization-induced conformational change, most likely inactivation. We attempted to simulate the disparate concentration-response relationships for the two effects of Na+o using a kinetic model that included Na+o site(s) affected by permeation and gating. While a model with only a single dynamic Na+o site was inadequate, a model with two distinct Na+o sites was sufficient to reproduce the data.