Abstract
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 CO(2) 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 CO(2). Six histidine residues are located in the C terminus. His-225, His-274, His-342, and His-354 were critical in CO(2) and pH sensing. Mutation of either of them reduced CO(2) and pH sensitivities by 20-50% and approximately 0.2 pH units, respectively. Simultaneous mutations of all of them eliminated the CO(2) 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 CO(2) and pH sensing is unclear, because substitutions of this residue with either a neutral, negative, or positive amino acid did not produce any functional channel. These results therefore indicate that histidine residues contribute to the sensitivity of the ROMK1 channel to hypercapnia and intracellular acidosis.
Highlights
ROMK channels (Kir1.1 and Kir1.2), members in the inward rectifier Kϩ channel family, were first cloned in the kidney and have later been found in several organs including the central nervous system [1, 2]
Our results indicate that His-225, His-274, His-342, and His-354 play a role in CO2 and pH sensing of the ROMK1 channel
Histidine Residues in the N Terminus—Our results have suggested that ROMK1 is inhibited during hypercapnia, and this is likely to be mediated by a decrease in intracellular pH
Summary
ROMK channels (Kir1.1 and Kir1.2), members in the inward rectifier Kϩ channel family, were first cloned in the kidney and have later been found in several organs including the central nervous system [1, 2]. A nontitratable threonine residue at position 51 of ROMK2 is involved in pH sensing [8] Mutation of this threonine to a negatively charged residue enhances pH sensitivity, whereas switching it to a positive amino acid decreases the pH sensitivity [8]. Another residue related to pH sensing is histidine 206. Two cysteine residues (Cys-49 and Cys-308) are critical players in the recovery, they do not affect the channel inhibition per se [11] These observations clearly demonstrate that multiple residues are involved in proton sensing in ROMK proteins, which one of them is the protonbinding site remains to be known. Our results indicate that His-225, His-274, His-342, and His-354 play a role in CO2 and pH sensing of the ROMK1 channel
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