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...

Voltage-dependent K+ channels can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel’s selectivity filter. C-type inactivation may involve movements of transmembrane voltage sensor domains, although the mechanisms underlying this form of inactivation may be heterogeneous and are often unclear. Here, we report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K+ channel that lacks a canonical voltage sensor and may thus provide a reduced system to inform on mechanism. In single-channel recordings, we observe that Po decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca2+ or Ba2+, suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K+] (47 mV per 10-fold increase in [K+]), suggesting that K+ binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K+ ≈ Rb+ > Cs+ > Na+ > Li+ ≈ NMG+. Selectivity of the stabilizing site is weaker than that of sites that determine permeability of these ions, suggesting that the site may lie toward the external end of the MthK selectivity filter. We could describe MthK gating over a wide range of positive voltages and external [K+] using kinetic schemes in which the open-conductive state is stabilized by K+ binding to a site that is not deep within the electric field, with the voltage dependence of inactivation arising from both voltage-dependent K+ dissociation and transitions between nonconducting (inactivated) states. These results provide a quantitative working hypothesis for voltage-dependent, K+-sensitive inactivation gating, a property that may be common to other K+ channels.

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did not alter the structure of the selectivity filter but did eliminate ion binding specifically to the S2 site. We introduced an equivalent ester substitution at the 2' position in the selectivity filter of the voltage-gated K þ channel KvAP and found that this substitution dramatically slowed inactivation, similar to the effect observed in the KcsA channel. Our results suggest that ion occupancy at the S2 site is necessary for C-type inactivation in K þ channels.

Polyamines such as spermidine and spermine are found in nearly all cells, at concentrations ranging up to 0.5 mM. These cations are endogenous regulators of cellular K+ efflux, binding tightly in the pores of inwardly rectifying K+ (Kir) channels in a voltage-dependent manner. Although the voltage dependence of Kir channel polyamine blockade is thought to arise at least partially from the energetically coupled movements of polyamine and K+ ions through the pore, the nature of physical interactions between these molecules is unclear. Here we analyze the polyamine-blocking mechanism in the model K+ channel MthK, using a combination of electrophysiology and computation. Spermidine (SPD3+) and spermine (SPM4+) each blocked current through MthK channels in a voltage-dependent manner, and blockade by these polyamines was described by a three-state kinetic scheme over a wide range of polyamine concentrations. In the context of the scheme, both SPD3+ and SPM4+ access a blocking site with similar effective gating valences (0.84 ± 0.03 e0 for SPD3+ and 0.99 ± 0.04 e0 for SPM4+), whereas SPM4+ binds in the blocked state with an ∼20-fold higher affinity than SPD3+ (Kd = 28.1 ± 3.1 µM for SPD3+ and 1.28 ± 0.20 µM for SPM4+), consistent with a free energy difference of 1.8 kcal/mol. Molecular simulations of the MthK pore in complex with either SPD3+ or SPM4+ are consistent with the leading amine interacting with the hydroxyl groups of T59, at the selectivity filter threshold, with access to this site governed by outward movement of K+ ions. These coupled movements can account for a large fraction of the voltage dependence of blockade. In contrast, differences in binding energetics between SPD3+ and SPM4+ may arise from distinct electrostatic interactions between the polyamines and carboxylate oxygens on the side chains of E92 and E96, located in the pore-lining helix.