Evaluating protein complexes between human aquaporin and calmodulin using biomolecular fluorescence complementation

Area of Science:

• Membrane biophysics
• Protein-protein interactions
• Molecular biology

Background:

• Aquaporins (AQPs) facilitate transmembrane water and solute transport.
• Calmodulin (CaM) is a key calcium-binding protein regulating various cellular processes.
• CaM's role in modulating specific eukaryotic aquaporin function is increasingly recognized.

Purpose of the Study:

• To systematically investigate interactions between all 13 human aquaporin (hAQP) homologues and calmodulin (CaM) in vivo.
• To identify novel CaM-binding hAQPs and understand their regulation.
• To establish a high-throughput screening method for membrane protein interactions.

Main Methods:

• Recombinant expression of 13 human aquaporins in yeast Saccharomyces cerevisiae.
• High-throughput screening using flow cytometry and microscopy.
• Bimolecular Fluorescence Complementation (BiFC) assays to detect protein complex formation.
• In silico analysis of potential CaM binding sites.

Main Results:

• Validated CaM binding to hAQP1 and hAQP4, building on known hAQP0-CaM interactions.
• Observed significant CaM interaction signals for hAQP8 and hAQP9, independent of their production levels.
• Demonstrated a robust yeast-based system for screening CaM-aquaporin interactions.

Conclusions:

• Calmodulin interacts with a subset of human aquaporins, including novel interactions with hAQP8 and hAQP9.
• The developed Bimolecular Fluorescence Complementation (BiFC) system in yeast is effective for discovering membrane protein interactions.
• These findings provide new insights into the regulatory mechanisms of human aquaporins by calmodulin.