Interactions of Cations with the Cytoplasmic Pores of Inward Rectifier K + Channels in the Closed State

Abstract

Ion channels gate at membrane-embedded domains by changing their conformation along the ion conduction pathway. Inward rectifier K + (Kir) channels possess a unique extramembrane cytoplasmic domain that extends this pathway. However, the relevance and contribution of this domain to ion permeation remain unclear. By qualitative x-ray crystallographic analysis, we found that the pore in the cytoplasmic domain of Kir3.2 binds cations in a valency-dependent manner and does not allow the displacement of Mg 2+ by monovalent cations or spermine. Electrophysiological analyses revealed that the cytoplasmic pore of Kir3.2 selectively binds positively charged molecules and has a higher affinity for Mg 2+ when it has a low probability of being open. The selective blocking of chemical modification of the side chain of pore-facing residues byMg 2+ indicates that the mode of binding of Mg 2+ is likely to be similar to that observed in the crystal structure. These results indicate that the Kir3.2 crystal structure has a closed conformation with a negative electrostatic field potential at the cytoplasmic pore, the potential of which may be controlled by conformational changes in the cytoplasmic domain to regulate ion diffusion along the pore. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.