Transmembrane allosteric coupling of the gates in a potassium channel

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.Potassium 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 inactivated state. For these reasons we tested this correspondence using NMR experiments. If the low K⁺ state is similar to the inactivated state of KcsA achieved by lowering the pH, it is expected that structural changes indicative of channel opening observed at low [K⁺] would occur not only in the selectivity filter but also in the pH gate and the hinge region. However, some studies imply that these two gates might be uncoupled or weakly coupled. For example, X-ray crystallographic studies of KcsA, where K⁺ sensitivity was largely isolated to the selectivity filter (3). In this work, we asked whether, by contrast, full-length wild-type KcsA (160 aa) reconstituted into hydrated lipid bilayers exhibits global structural changes upon ion expulsion suggestive of channel opening. To accomplish this, nearly complete ¹³C and ¹⁵N chemical shift assignments were obtained for the transmembrane and loop regions from four-dimensional (4D) solid-state nuclear magnetic resonance (SSNMR) (27), providing numerous reporters for conformational change during ion binding. In low [K⁺] ion conditions at neutral pH, not only does KcsA expel the K⁺ ions from the inner selectivity filter sites, but the channel also exhibits chemical shift perturbations at the pH gate and the hinge of the inner transmembrane helix, suggesting features akin to the inhibited state that is present at low pH and high [K⁺]. That these two distinct conditions result in a nearly identical state of the channel offers strong evidence for transmembrane allostery in the inactivation process.