Involvement of Histidine Residues in Proton Sensing of ROMK1 Channel

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 ϳ0.2 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.ROMK channels (Kir1.1 and Kir1.2), members in the inward rectifier K ϩ channel family, were first cloned in the kidney and have later been found in several organs including the central nervous system (1, 2). These K ϩ channels are believed to control K ϩ secretion in the renal tubular cells and membrane potential in excitable cells (3,4). ROMK channels have a relatively weak inward rectification allowing significant K ϩ currents in the outward direction and are subject to extensive modulations by second messengers, protein kinases, phospholipids, ATP, and other nucleotides (5). Another important modulator of the ROMK channels is proton. A decrease in intracellular pH strongly inhibits these channels (6 -11).Proton sensing in ROMK channels requires multiple sites or residues in the channel protein to interact with protons. A lysine residue (Lys-80 in ROMK1 or Lys-61 in ROMK2) in the N terminus of channel proteins plays a critical part in the channel sensitivity to intracellular pH. Mutation of this residue to methionine greatly reduces the pH sensitivity (8, 10). However, lysine is not titratable at a physiological pH range. Thus, how this lysine residue works in pH sensing is not fully understood. A nontitratable threonine residue at position 51 of ROMK2 is also 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. Mutation of this residue to glycine enhances channel sensitivity to pH (10). Supporting multiple interaction...

Requirement of Multiple Protein Domains and Residues for GatingKATP Channels by Intracellular pH

“…In Kir1.1, several residues in the N and C termini are known to be involved in the pH sensitivity (23,27,38,39). In Kir2.3, proton sensing relies on an interaction of both N and C termini at near the membrane-spanning regions, whereas proton sensors are likely to be located in more distal areas of the C terminus (34).…”

“…Kir6.2⌬C36 that expresses functional channels without the SUR subunit and retains fair ATP sensitivity (22). Several chimerical channels were generated based on peptide sequences of Kir6.2 and Kir1.1, a Kir channel that is inhibited by intracellular acidosis (23)(24)(25) and has been shown to express functional channels in its chimeras with Kir6.2 (26). These chimeras were studied in whole-cell recording using hypercapnia, a condition that does not cause channel rundown (21).…”

Rare independent mutations in renal salt handling genes contribute to blood pressure variation

“…We first identified well-documented disease causing mutations which had been shown biochemically to cause loss of function of the encoded cotransporter/ channel [18][19][20][21][22] . This identified 10 different biochemically proven loss of function mutations found in 23 subjects (Table 1).…”

Acute pH-dependent Regulation of AE2-mediated Anion Exchange Involves Discrete Local Surfaces of the NH2-terminal Cytoplasmic Domain

“…sensitivity, individual mutation of four of the COOH-terminal tail His residues attenuates pH sensitivity, and mutation of all four residues nearly completely abolishes it (27).…”

“…Histidine-Exofacial His protonation controls the pHdependent activity of many ion channel polypeptides (27)(28)(29).…”