Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel

摘要

MthK is a Ca²⁺-gated K⁺ channel whose activity is inhibited by cytoplasmic H⁺. To determine possible mechanisms underlying the channel’s proton sensitivity and the relation between H⁺ inhibition and Ca²⁺-dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca²⁺] (ranging from nominally 0 to 30 mM) at a given [H⁺], in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca²⁺] and open probability (Po), with a mean Hill coefficient (nH) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor nH changed significantly as a function of [H⁺] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca²⁺ was not H⁺ sensitive over pH ranging from 7.3 to 9.0. However, increasing [H⁺] raised the EC50 for Ca²⁺ activation by ∼4.7-fold per tenfold increase in [H⁺], displaying a linear relation between log(EC50) and log([H⁺]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H⁺ binding does not directly modulate either the channel’s closed–open equilibrium or the allosteric coupling between Ca²⁺ binding and channel opening. We can account for the Ca²⁺ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca²⁺ allosterically activates MthK, whereas H⁺ opposes activation by destabilizing the binding of Ca²⁺.