K + efflux through K + channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel's selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K + channel. Either Mg 2+ or Ca 2+ can reduce K + efflux through MthK channels. However, Ca 2+ , but not Mg 2+ , can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca 2+ ions can partially dehydrate, enabling selective accessibility of Ca 2+ to a site at the entry to the selectivity filter. Ca 2+ binding at the site interacts with K + ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.calcium | gating | permeation | dynamics | energetics P otassium (K + ) channels are activated and opened by a variety of stimuli, including ligand binding and transmembrane voltage, to enable K + efflux and thus, modulate physiological processes related to electrical excitability, such as regulation of action potential firing, smooth muscle contraction, and hormone secretion (1). In addition, many K + channels are further controlled by a gating phenomenon known as C-type inactivation, in which K + conduction is stopped, despite the continued presence of an activating stimulus (2). The mechanisms underlying C-type inactivation in voltage-gated K + channels (Kv channels) are linked to both intracellular and extracellular permeant ion concentrations, and several lines of evidence have suggested that Ctype inactivation is associated with a conformational change near the external mouth of the K + channel pore (i.e., at the canonical K + channel selectivity filter) (3-11).In Shaker Kv channels, C-type inactivation is known to be enhanced and recovery from inactivation is slowed by impermeant cations accessing the cytoplasmic side of the channel (5, 6, 10). Enhancement of inactivation by these cations suggests a working hypothesis, in which the impermeant ion prevents refilling of the selectivity filter with K + (6). Thus, K + presumably dissociates from the filter to the external solution, and this vacancy leaves the filter susceptible to a conformational change that underlies the nonconducting, inactivated state. However, the physical basis for the relation between ion movements and C-type inactivation as well as the structural underpinnings of the mechanism remain unclear.Here, we use divalent metal cations (Mg 2+ , Ca 2+ , and Sr 2+ ) as probes of inactivation mechanisms in MthK, a model K + channel of known structure (Fig. 1) (12-14). Specifically, we analyze conduction and gating of single MthK channels by electrophysiology combined with analysis of ion and protein movements by molecular simulation. Our el...