Initial steps of inactivation at the K ⁺ channel selectivity filter

Abstract

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...

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

“…These may reduce K + affinity and increase ion permeation, leading to a conducting (open) state; after opening, however, these movements may be coupled to further structural changes at the selectivity filter that block permeation, leading to an inactivated state (Figure 2B) [185–188]. These observations are consistent with combined electrophysiological and computational studies in MthK, which show that reduction in extracellular K + can lead to K + dissociation, followed by structural changes in the selectivity filter to a non-conducting state [108, 164]. Thus, a combination of structural, functional, and computational studies point to a mechanism in which movement of the pore-lining helices of K + channels can be structurally and energetically coupled with gating at the selectivity filter.…”

“…Although permeation and gating have been largely studied as separate phenomena, it has been known since the seminal work of Clay Armstrong that permeant ions can strongly influence channel gating; indeed, it has become increasingly clear in K + channels that these processes are inextricably linked [165–170]. The conducting state of the selectivity filter, a narrow region of the K + channel pore that can discriminate between K + and other physiologically abundant ions, is stabilized through interactions with K + [108, 164, 165]. Through these and other interactions, the selectivity filter may itself act as a molecular gate to open and close the channel [163, 164, 171, 172].…”

“…The conducting state of the selectivity filter, a narrow region of the K + channel pore that can discriminate between K + and other physiologically abundant ions, is stabilized through interactions with K + [108, 164, 165]. Through these and other interactions, the selectivity filter may itself act as a molecular gate to open and close the channel [163, 164, 171, 172]. …”

Atomic mutagenesis in ion channels with engineered stoichiometry

“…In a physiological setting, this mechanism represents a form of activity-dependent macromolecular memory that can alter action-potential firing rates (Aldrich et al, 1979; Roeper et al, 1997). The basis for C-type inactivation has been investigated with functional, structural and computational approaches, which have produced a consensus mechanism whereby channels become non-conducting due to local perturbations in the selectivity filter (Yellen et al, 1994; Baukrowitz and Yellen, 1995; Kiss and Korn, 1998; Perozo et al, 1998; Roux and MacKinnon, 1999; Bernèche and Roux, 2001; Cordero-Morales et al, 2007; Panyi and Deutsch, 2007; Cuello et al, 2010; Ostmeyer et al, 2013; Thomson et al, 2014). However, the nature of the conformational change and role of specific residues remain an area of active experimentation and debate (Devaraneni et al, 2013; Hoshi and Armstrong, 2013).…”

Unambiguous observation of blocked states reveals altered, blocker-induced, cardiac ryanodine receptor gating

“…While ionic flux through the pore is controlled by the gates, the ion occupancy itself was found to have an effect on channel gating behaviour by bringing about subtle conformational changes in the SF region, a phenomenon that has been the focus of several recent studies 6 7 8 . The role of ion occupancy in SF-mediated gating has been investigated by altering the concentration of ions as well as by replacing the permeant ion (usually K + ) with other monovalent and divalent cations to bring about changes in pore conformation and affect gating 9 10 . Large tetra-alkyl ammonium cations (TAAs), which are pore blockers in K + channels, have also been employed as probes to study the effects of resulting perturbations in ion occupancy on channel gating 11 12 13 14 .…”