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Updated: Jun 26, 2025

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Published on: July 28, 2014
Molecular and Functional Profiling of Gαi as an Intracellular pH Sensor
Ajit Prakash1, Zijian Li1, Venkata R Chirasani1
1Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Intracellular pH changes alter the structure and stability of Gαi proteins, impacting G-protein-coupled receptor (GPCR) signaling. This study reveals a pH-sensing network in Gαi, offering new therapeutic targets for diseases involving GPCR pathways.
Area of Science:
- Molecular and Cellular Biology
- Biochemistry
- Physiology
Background:
- Heterotrimeric G proteins (Gα, Gβ, Gγ) are crucial downstream effectors of G-protein-coupled receptors (GPCRs).
- Previous studies suggested Gαi and its yeast homolog Gpa1 act as intracellular pH sensors, but the mechanism was unclear.
Purpose of the Study:
- To identify the pH-sensing network within Gαi.
- To investigate the impact of pH modulation on Gαi structure, stability, and function.
- To explore therapeutic strategies targeting pH-dependent Gαi activity.
Main Methods:
- Structural and stability analysis of Gαi-GDP under varying physiological pH conditions.
- Cellular assays in HEK293 cells to assess Gαi-Gβγ release.
- Site-directed mutagenesis to generate low pH mimetics targeting key residues.
Main Results:
- Physiological pH changes significantly alter Gαi-GDP structure and stability, inducing a disorder-to-order transition between pH 6.8 and 7.5.
- Intracellular pH modulation in HEK293 cells regulates Gαi-Gβγ subunit release.
- Engineered low pH mimetics effectively attenuate Gαi-Gβγ release.
Conclusions:
- Gαi possesses a pH-sensing network, and pH-dependent structural changes modulate Gβγ subunit dissociation.
- These findings reveal a novel mechanism by which intracellular pH influences GPCR signaling pathways.
- Targeting the Gαi pH-sensing network offers potential therapeutic avenues for diseases linked to aberrant GPCR signaling.
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