Involvement of histidine residues in proton sensing of ROMK1 channel

ROMK channels are inhibited by intracellular acidification, This pH sensitivity is related to several amino acid residues in the channel proteins such as Lys-61, Thr-51, and His-206 (in ROMK2), Unlike all other amino acids, histidine is titratable at pH 6-7 carrying a positive charge below pH 6, To test the hypothesis that certain histidine residues are engaged in CO2 and pH sensing of ROMK1, we performed experiments by systematic mutations of all histidine residues in the channel using the site-directed mutagenesis, There are two histidine residues in the N terminus. Mutations of His-23, His-31, or both together did not affect channel sensitivity to CO2. Six histidine residues are located in the C terminus. His-225, His-274, His-342, and His-354 were critical in CO2 and pH sensing. Mutation of either of them reduced CO2 and pH sensitivities by 20-50% and similar to 0.2 pH units, respectively. Simultaneous mutations of all of them eliminated the CO2 sensitivity and caused this mutant channel to respond to only extremely acidic pH, Similar mutations of His-280 had no effect. The role of His-270 in CO2 and pH sensing is unclear, because substitutions of this residue with either a neutral, negative, or positive amino acid did not produce any fumctional channel. These results therefore indicate that histidine residues contribute to the sensitivity of the ROMK1 channel to hypercapnia and intracellular acidosis.