Gating of inward rectifier K+ channels by proton-mediated interactions of N- and C-terminal domains

Ion channels play an important role in cellular functions, and specific cel lular activity can be produced by gating them. One important gating mechani sm is produced by intra- or extracellular ligands, Although the ligand-medi ated channel gating is an important cellular process, the relationship betw een ligand binding and channel gating is not well understood, It is possibl e that ligands are involved in the interactions of different protein domain s of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results s howed that hypercapnia or intracellular acidification strongly inhibited th ese channels. This effect relied on both the N and C termini, The CO2/pH se nsitivities were abolished or compromised when one of the intracellular ter mini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding w as weak at pH 7.4, stronger binding was seen at pH 6.6, Two short sequences in the N and C termini were found to be critical for the N/C-terminal inte raction. Interestingly, there was no titratable residue in these motifs, To identify the potential protonation sites, we systematically mutated most h istidine residues in the intracellular N and C termini, We found that mutat ions of several histidine residues in the C but not the N terminus had a ma jor effect on channel sensitivities to CO2 and pH(i). These results suggest that at acidic pH, protons appear to interact with the C-terminal histidin e residues and present the C terminus to the N terminus. Consequentially, t hese two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K+ channel gating is d emonstrated, which involves the N- and C-terminal interaction with protons as the mediator.