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Structural and functional analysis of the putative pH sensor in the Kir1.1 (ROMK) potassium channel.

ABSTRACT: The pH-sensitive renal potassium channel Kir1.1 is important for K+ homeostasis. Disruption of the pH-sensing mechanism causes type II Bartter syndrome. The pH sensor is thought to be an anomalously titrated lysine residue (K80) that interacts with two arginine residues as part of an 'RKR triad'. We show that a Kir1.1 orthologue from Fugu rubripes lacks this lysine and yet is still highly pH sensitive, indicating that K80 is not the H+ sensor. Instead, K80 functionally interacts with A177 on transmembrane domain 2 at the 'helix-bundle crossing' and controls the ability of pH-dependent conformational changes to induce pore closure. Although not required for pH inhibition, K80 is indispensable for the coupling of pH gating to the extracellular K+ concentration, explaining its conservation in most Kir1.1 orthologues. Furthermore, we demonstrate that instead of interacting with K80, the RKR arginine residues form highly conserved inter- and intra-subunit interactions that are important for Kir channel gating and influence pH sensitivity indirectly.

SUBMITTER: Rapedius M

PROVIDER: S-EPMC1479598 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Structural and functional analysis of the putative pH sensor in the Kir1.1 (ROMK) potassium channel.

Rapedius Markus M Haider Shozeb S Browne Katharine F KF Shang Lijun L Sansom Mark S P MS Baukrowitz Thomas T Tucker Stephen J SJ

EMBO reports 20060421 6

The pH-sensitive renal potassium channel Kir1.1 is important for K+ homeostasis. Disruption of the pH-sensing mechanism causes type II Bartter syndrome. The pH sensor is thought to be an anomalously titrated lysine residue (K80) that interacts with two arginine residues as part of an 'RKR triad'. We show that a Kir1.1 orthologue from Fugu rubripes lacks this lysine and yet is still highly pH sensitive, indicating that K80 is not the H+ sensor. Instead, K80 functionally interacts with A177 on tra ...[more]

PMID: 16641935

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Project description:Gating of inward rectifier Kir1.1 potassium channels by internal pH is believed to occur when large hydrophobic leucines, on each of the four subunits, obstruct the permeation path at the cytoplasmic end of the inner transmembrane helices (TM2). In this study, we examined whether closure of the channel at this point involves bending of the inner helix at one or both of two highly conserved glycine residues (corresponding to G134 and G143 in KirBac1.1) that have been proposed as putative "gating hinges" for potassium channels. Replacement of these conserved inner helical glycines by less flexible alanines did not abolish gating but shifted the apparent pKa from 6.6 +/- 0.01 (wild-type) to 7.1 +/- 0.01 for G157A-Kir1.1b, and to 7.3 +/- 0.01 for G148A-Kir1.1b. When both glycines were mutated the effect was additive, shifting the pKa by 1.2 pH units to 7.8 +/- 0.04 for the double mutant: G157A+G148A. At this pKa, the double mutant would remain completely closed under physiological conditions. In contrast, when the glycine at G148 was replaced by a proline, the pKa was shifted in the opposite direction from 6.6 +/- 0.01 (wild-type) to 5.7 +/- 0.01 for G148P. Although conserved glycines at G148 and G157 made it significantly easier to open the channel, they were not an absolute requirement for pH gating in Kir1.1. In addition, none of the glycine mutants produced more than small changes in either the cell-attached or excised single-channel kinetics which, in this channel, argues against changes in the selectivity filter. The putative pH sensor at K61-Kir1.1b, (equivalent to K80-Kir1.1a) was also examined. Mutation of this lysine to an untitratable methionine did not abolish pH gating, but shifted the pKa into an acid range from 6.6 +/- 0.01 to 5.4 +/- 0.04, similar to pH gating in Kir2.1. Hence K61-Kir1.1b cannot function as the exclusive pH sensor for the channel, although it may act as one of multiple pH sensors, or as a link between a cytoplasmic sensor and the channel gate. K61-Kir1.1b also interacted differently with the two glycine mutations. Gating of the double mutant: K61M+G148A was indistinguishable from K61M alone, whereas gating of K61M+G157A was midway between the alkaline pKa of G157A and the acid pKa of K61M. Finally, closure of ROMK, G148A, G157A, and K61M all required the same L160-Kir1.1b residue at the cytoplasmic end of the inner transmembrane helix. Hence in wild-type and mutant channels, closure occurs by steric occlusion of the permeation path by four leucine side chains (L160-Kir1.1b) at the helix bundle crossing. This is facilitated by the conserved glycines on TM2, but pH gating in Kir1.1 does not absolutely require glycine hinges in this region.

Dataset Information

Structural and functional analysis of the putative pH sensor in the Kir1.1 (ROMK) potassium channel.

Publications

Structural and functional analysis of the putative pH sensor in the Kir1.1 (ROMK) potassium channel.

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