Random mutagenesis screening indicates the absence of a separate H(+)-sensor in the pH-sensitive Kir channels

Area of Science:

• Molecular Biology
• Biophysics
• Ion Channel Physiology

Background:

• Inwardly-rectifying (Kir) potassium channels, including Kir1.1, Kir4.1, and Kir4.2, are inhibited by intracellular H+ within physiological ranges.
• The precise molecular mechanism and identity of the pH-sensor in these channels remain elusive despite extensive research.
• Understanding intracellular pH regulation of Kir channels is crucial for various physiological processes.

Purpose of the Study:

• To identify mutations in Kir1.1 channels that alter sensitivity to intracellular pH.
• To elucidate the molecular basis of pH sensing in inwardly-rectifying potassium channels.
• To investigate the role of specific residues in channel gating and pH response.

Main Methods:

• Utilized a potassium (K+)-auxotrophic strain of Saccharomyces cerevisiae with an acidic intracellular environment.
• Performed an unbiased genetic screen to identify Kir1.1 mutants with impaired pH-sensitivity.
• Characterized identified mutations, including K80M and a novel S172T mutation in the second transmembrane domain (TM2).

Main Results:

• Identified a novel S172T mutation in TM2 that significantly reduces pH-sensitivity by destabilizing the closed-state.
• Confirmed the previously identified K80M mutation's effect on pH-sensitivity.
• Failed to identify mutations that completely abolish pH-sensitivity or act as unique H+ sensors.

Conclusions:

• Propose a model where pH sensing is an intrinsic gating mechanism inherent to all Kir channels, not exclusive to pH-sensitive subtypes.
• Hypothesize that mutations disrupting the pH-sensor would increase, not decrease, pH-sensitivity, explaining the difficulty in identifying specific sensor residues.
• The findings necessitate a re-evaluation of future studies on Kir channel pH-sensitivity and the identification of pH-sensing residues.