Gating and inward rectifying properties of the MthK K⁺ channel with and without the gating ring

In MthK, a Ca²⁺-gated K⁺ channel from Methanobacterium thermoautotrophicum, eight cytoplasmic RCK domains form an octameric gating ring that controls the intracellular gate of the ion conduction pore. The binding of Ca²⁺ ions to the RCK domains alters the conformation of the gating ring, thereby opening the gate. In the present study, we examined the Ca²⁺- and pH-regulated gating and the rectifying conduction properties of MthK at the single-channel level. The open probability (P_o) of MthK exhibits a sigmoidal relationship with intracellular [Ca²⁺], and a Hill coefficient >1 is required to describe the dependence of P_o on [Ca²⁺], suggesting cooperative Ca ²⁺ activation of the channel. Additionally, intracellular Ca ²⁺ also blocks the MthK pore in a voltage-dependent manner, rendering an apparently inwardly rectifying I-V relation. Intracellular pH has a dual effect on MthK gating. Below pH 7.5, the channel becomes insensitive to Ca ²⁺. This occurs because the gating ring is structurally unstable at this pH and tends to disassemble (Ye, S., Y. Li, L. Chen, and Y. Jiang. 2006. Cell. 126:1161-1173). In contrast, above pH 7.5, a further increase in pH shifts the P_o-[Ca²⁺] relation towards a lower Ca²⁺ concentration, augments P_o at saturating [Ca²⁺], and activates the channel even in the absence of Ca²⁺. Channel activity is marked by bursts of rapid openings and closings separated by relatively longer interburst closings. The duration of interburst closing and the burst length are highly Ca²⁺ and pH dependent, whereas the kinetics of intraburst events is Ca²⁺ and pH independent. The rapid intraburst openings and closings are also observed with the isolated MthK pore lacking the attached intracellular gating ring. The fast kinetic events, independent of both Ca²⁺ and pH, therefore appear to be determined by processes occurring within the ion conduction pore, whereas the slow events reflect the gating process controlled by Ca²⁺ and pH through the gating ring.