Abstract
Inwardly rectifying potassium (Kir) channels play a critical role in stabilizing the membrane potential, thus controlling numerous physiological phenomena in multiple tissues. Channel conductance is activated by cytoplasmic modulators that open the channel at the 'helix bundle crossing' (HBC), formed by the coming together of the M2 helices from each of the four subunits, at the cytoplasmic end of the transmembrane pore. We introduced a negative charge at the bundle crossing region (G178D) in classical inward rectifier Kir2.2 channel subunits that forces channel opening, allowing pore wetting and free movement of permeant ions between the cytoplasm and the inner cavity. Single-channel recordings reveal a striking pH-dependent subconductance behavior in G178D (or G178E and equivalent Kir2.1[G177E]) mutant channels that reflects individual subunit events. These subconductance levels are well resolved temporally and occur independently, with no evidence of cooperativity. Decreasing cytoplasmic pH shifts the probability towards lower conductance levels, and molecular dynamics simulations show how protonation of Kir2.2[G178D] and, additionally, the rectification controller (D173) pore-lining residues leads to changes in pore solvation, K+ ion occupancy, and ultimately K+ conductance. While subconductance gating has long been discussed, resolution and explanation have been lacking. The present data reveals how individual protonation events change the electrostatic microenvironment of the pore, resulting in distinct, uncoordinated, and relatively long-lasting conductance states, which depend on levels of ion pooling in the pore and the maintenance of pore wetting. Gating and conductance are classically understood as separate processes in ion channels. The remarkable sub-state gating behavior of these channels reveals how intimately connected 'gating' and 'conductance' are in reality.