Abstract
Transmembrane allosteric coupling in C-type inactivation of a potassium channel is analyzed using NMR. Activation of KcsA is initiated by a decrease in pH on the intracellular side of the channel. Numerous studies suggest that the selectivity filter is subsequently allosterically affected by opening and that a conformational switch and loss of affinity for potassium ions is the central step of C-type inactivation. We tested this hypothesis using an open pH gate KcsA mutant (H25R/E118A) which is constitutively active and lacks pH-dependent behavior of the activation gate. We use Solid State NMR measurements of this open pH gate mutant at neutral pH in hydrated bilayers to probe the potassium ion concentration dependence. The potassium ion binding affinity of the selectivity filter is 81 mM, about 4 orders of magnitude weaker than for wild type KcsA at the same pH, but similar to the value for wild type KcsA when activated at low pH. These results underscore the fact that ion affinity at the selectivity function is very much weaker in the open, activated state as compared to the closed, deactivated resting state, and confirm the role of allosteric coupling in the delayed inactivation mechanism. The protonation state of an important glutamate residue (E120) in the pH sensor of the activation gate changes as a function of potassium concentration, indicating that this mutant also exhibits transmembrane allosteric coupling.
Footnotes
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