Engineering a human P2X2 receptor with altered ligand selectivity in yeast

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• P2X receptors are ligand-gated ion channels crucial in various biological processes.
• These receptors are found in many species but not naturally in yeast (Saccharomyces cerevisiae).
• Mammalian expression systems for ion channel research are often costly and time-consuming.

Purpose of the Study:

• To achieve the first recombinant expression and functional gating of a human P2X2 receptor in baker's yeast.
• To utilize yeast as a host for efficient genetic screening and engineering of P2X2 receptor mutants.
• To engineer P2X2 variants with altered ligand selectivity and gating properties.

Main Methods:

• Site saturation mutagenesis was performed on the P2X2 receptor within the yeast Saccharomyces cerevisiae.
• Deep mutational analysis was employed to identify key residues affecting receptor function.
• The engineered yeast system facilitated functional characterization of P2X2 receptor variants.

Main Results:

• The study successfully expressed and functionally gated the P2X2 receptor in yeast.
• A mutant P2X2 receptor (F303Y A304W) was engineered with altered selectivity for the ATP analog AMP-PNP.
• The F303Y A304W variant exhibited over 100-fold increased intracellular calcium amplitudes and reduced desensitization with AMP-PNP.

Conclusions:

• Baker's yeast provides a scalable and cost-effective platform for ion channel characterization and engineering.
• The engineered P2X2 receptor variant demonstrates potential for chemogenetic cellular control applications.
• The A304W mutation's destabilization of the desensitized state offers mechanistic insights into receptor gating with suboptimal agonists.