A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating

Abstract

ROMK channels play a key role in overall K balance by controlling K secretion across the apical membrane of mammalian cortical collecting tubule. In contrast to the family of strong inward rectifiers (IRKs), ROMK channels are markedly sensitive to intracellular pH. Using Xenopus oocytes, we have confirmed this pH sensitivity at both the single-channel and whole cell level. Reduction of oocyte pH from 6.8 to 6.4 (using a permeant acetate buffer) reduced channel open probability from 0.76 ± 0.02 to near zero ( n = 8), without altering single-channel conductance. This was due to the appearance of a long-lived closed state at low internal pH. We have confirmed that a lysine residue (K61 on ROMK2; K80 on ROMK1), NH2 terminal to the first putative transmembrane segment (M1), is primarily responsible for conferring a steep pH sensitivity to ROMK (B. Fakler, J. Schultz, J. Yang, U. Schulte, U. Bråandle, H. P. Zenner, L. Y. Jan, and J. P. Ruppersberg. EMBO J. 15: 4093–4099, 1996). However, the apparent p K a of ROMK also depends on another residue in a highly conserved, mildly hydrophobic area: T51 on ROMK2 (T70 on ROMK1). Replacing this neutral threonine (T51) with a negatively charged glutamate shifted the apparent p K a for inward conductance from 6.5 ± 0.01 ( n = 8, wild type) to 7.0 ± 0.02 ( n = 5, T51E). On the other hand, replacing T51 with a positively charged lysine shifted the apparent p K a in the opposite direction, from 6.5 ± 0.01 ( n = 8, wild type) to 6.0 ± 0.02 ( n = 9, T51K). The opposite effects of the glutamate and lysine substitutions at position 51 (ROMK2) are consistent with a model in which T51 is physically close to K61 and alters either the local pH or the apparent p K a via an electrostatic mechanism. In addition to its effects on pH sensitivity, the mutation T51E also decreased single-channel conductance from 34.0 ± 1.0 pS ( n = 8, wild type) to 17.4 ± 1 pS ( n = 9, T51E), reversed the voltage gating of the channel, and significantly increased open-channel noise. These effects on single-channel currents suggest that the T51 residue, located in a mildly hydrophobic area of ROMK2, also interacts with the hydrophobic region of the permeation pathway.

Subunit–subunit interactions are critical for proton sensitivity of ROMK: Evidence in support of an intermolecular gating mechanism

“…2 A). Similar observations for ROMK1 and ROMK2 have been reported by others (8,10). The half-maximal inhibitory pH values, pH 0.5 , and Hill coefficients of the isoforms were not significantly different (Fig.…”

“…1) produced pH-insensitive channels (8,10,14); however, a more complete pH titration to 4 demonstrated that each of the three ROMK isoforms remained pH sensitive, with the N-terminal K3 M mutation ( Fig. 2 B and C).…”

Multiple targets of chemosensitive signaling in locus coeruleus neurons: role of K+ and Ca2+channels

“…pH i sensitivity has been observed in members of the K ir (12,39), the K Ca (31,49), and the K v (6) channel family. pH o sensitivity has been observed in members of the TASK channel family (5,22).…”

Identification of the Erythrocyte Rh Blood Group Glycoprotein as a Mammalian Ammonium Transporter

“…A strategy that has been well documented for acidifying oocytes uses acetate-buffered solutions. In the presence of 60 mM sodium acetate, the lipophilic protonated form of acetic acid crosses the membrane and dissociates, acidifying intracellular pH i (14,15). After 20 -25 min of incubation in acetate buffer the pH i reaches steady state and recovers on removal in ϳ15 min (14).…”

“…In the presence of 60 mM sodium acetate, the lipophilic protonated form of acetic acid crosses the membrane and dissociates, acidifying intracellular pH i (14,15). After 20 -25 min of incubation in acetate buffer the pH i reaches steady state and recovers on removal in ϳ15 min (14). Oocytes were preincubated for 25 min in acetate buffers adjusted from pH 6.1 to 7.4 and then moved to MA uptake buffer at pH 7.5 or 8.5, a treatment that is expected to lower pH i to approximately pH 5.9 -6.8 (14).…”