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 by Mg(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.
