Transmembrane allosteric coupling of the gates in a potassium channel

Abstract

It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98-103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.C-type inactivation | solid-state NMR | membrane protein | protein dynamics | chemical shift assignments P otassium channel activation and inactivation is fundamental to many physiological functions including muscle contraction and the generation of synaptic action potentials (1). KcsA is a 160-residue pH-activated homotetrameric K + channel isolated from the soil bacterium Streptomyces lividans (2, 3) with high sequence homology and functional similarity to mammalian potassium channels (4). It has provided an excellent model for studies of ion-conduction by X-ray crystallography (3, 5, 6), electrophysiology (7,8), and NMR (9-21). Like many potassium channels, it exhibits (4, 6, 22, 23) slow, spontaneous inactivation involving the residues near the extracellular selectivity filter subsequent to channel activation. Recent results from X-ray crystallography and molecular dynamics suggest that the gates are coupled and that inactivation is prompted by channel opening, mediated via a series of intrasubunit steric contacts involving F103 with T74, T75, and M96 and an intersubunit contact with the neighboring I100 side chain (4-6, 24, 25). In separate experiments, the extracellular gate has been observed to respond directly to ambient [K + ]: at high [K + ] it exists in a conductive form, and at low K + it collapses into a nonconductive state (3). Our NMR studies suggest that the low [K + ] state and the low pH inactivated state may be similar; this conclusion is supported by the effect of the mutation E71A and the pattern of chemical shift perturbations in the selectivity filter when the ion is depleted (9, 19). Meanwhile, X-ray crystallography studies suggest that mutants (E71A) unable to undergo inactivation are also unable to expel ions (26).An established similarity of the low pH and the low [K + ] states would clarify the importance of allosteric coupling and have the practical consequence that the well-behaved low K + state could serve as a useful structural proxy for the otherwise fleeting ...

Selective exclusion and selective binding both contribute to ion selectivity in KcsA, a model potassium channel

“…Such K + concentration-dependent conformational changes were also seen in NMR studies (9)(10)(11)(12)(13)(14). Low K + concentrations leads to a nonconductive conformation of the selectivity filter in which K + binds only at the extreme S1 and S4 sites, with an average occupancy of just one K + distributed between those two sites.…”

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

“…We could assign approximately half of the resonances in the spectrum in Figure 2 B based on 3D CαNH, Cα(CO)NH, CONH, and CO(Cα)NH experiments (Figure 2 F), supplemented with 3D NCαCX and 2D N(CO)CX spectra to identify residue types, and supported by available 13 C and 15 N chemical‐shift data 9c, 12. These assignments include a number of functionally essential structural elements that we can access here for the first time with 1 H detection.…”

“…However, the G43H N resonance, which is by far the broadest of all H N (0.44 ppm), is split into two signals with different relaxation decays (Figures 3 C, D). As the G43 chemical shift is sensitive to the filter ion‐binding mode,12 this splitting may hence also be related to the conformational flexibility of the filter. Intriguingly, unlike almost all other resonances, the G43H N resonance is much narrower (0.31 ppm) in situ, most likely because only one conformation is present (Figure 3 E).…”

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

“…These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1)(2)(3)(4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4)(5)(6)(7)(8)(9)(10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5)(6)(7)(8)(9)(10)(11)(12)(13)(14).…”

“…A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4)(5)(6)(7)(8)(9)(10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). However, flux-dependent inactivation occurs in Na + and Ca 2+ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7,(13)(14)(15)(16)(17)(18).…”