Role of conserved glycines in pH gating of Kir1.1 (ROMK).
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.
Department of Physiology and Biophysics, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Rd., North Chicago, IL 60064, USA. henry.sackin@rosalindfranklin.edu
Henry
H
Sackin
Mikheil
M
Nanazashvili
Lawrence G
LG
Palmer
Hui
H
Li
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D017124:Conserved Sequence; D005998:Glycine / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000737:chemistry* / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000502:physiology*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
C489461:Kir2.1 channel; D024661:Potassium Channels, Inwardly Rectifying; D005998:Glycine