A KcsA cytoplasmic pH-gate investigated in lipoprotein nanodiscs.

The bacterial potassium channel KcsA is gated by pH, opening for conduction under acidic conditions. Molecular determinants responsible for this effect have been identified at the extracellular selectivity filter, at the membrane-cytoplasm interface (TM2-gate), and in the cytoplasmic C-terminal domain (CTD), an amphiphilic four-helix bundle mediated by hydrophobic and electrostatic interactions. Here we employ nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) to provide a structural view of the pH-induced open-to-closed CTD transition. KcsA was embedded in lipoprotein nanodiscs (LPNs), selectively methyl-protonated at Leu/Val residues to allow observation of both states by NMR, and spin-labeled for purposes of EPR studies. We observed a pH-induced structural change between an associated structured CTD at neutral pH, and a dissociated flexible CTD at acidic pH, with a transition in the 5.0-5.5 range, consistent with a stabilization of the CTD by channel architecture. A double mutant constitutively open at the TM2-gate exhibited reduced stability of associated CTD, as indicated by weaker spin-spin interactions, a shift to higher transition pH values, and a 10-fold reduction in the population of the associated 'closed' channels. We thus extend findings for isolated CTD-derived peptides to full-length KcsA and establish a contribution of the CTD to KcsA pH-controlled gating, which exhibits a strong correlation with the state of the proximal TM2 gate.