Mutagenesis studies of TRPV1 subunit interfaces informed by genomic variant analysis

Area of Science:

• Molecular Biology
• Structural Biology
• Ion Channel Physiology

Background:

• Protein structure and mutagenesis are key to understanding ion channel mechanisms.
• Selecting mutagenesis sites can be challenging, necessitating refined approaches.
• Human population genomic data offers a novel resource for guiding mutagenesis strategies.

Purpose of the Study:

• To investigate the utility of human population genomic data in refining mutagenesis site selection for TRPV1.
• To identify and characterize critical residues at TRPV1 subunit interfaces using structural and genomic data.
• To elucidate the role of specific electrostatic interactions in TRPV1 channel gating and function.

Main Methods:

• Analysis of TRPV1 missense variants in gnomAD 2.1.1 human population data.
• Identification of potential electrostatic networks in the TRPV1 structure (PDB: 7LQY).
• Site-directed mutagenesis of identified residues (K155, K160, E761, D762) and functional characterization using Ca2+ imaging and electrophysiology.

Main Results:

• Missense variant analysis revealed fewer variants in buried ankyrin repeat domain residues and more in transmembrane segments.
• No gnomAD variants were found for the identified K-KED interface residues (K155, K160, E761, D762).
• Mutagenesis studies confirmed the importance of K-KED residues, but electrostatic interactions were not essential; charge reversals altered channel function and baseline currents, with a double mutant partially restoring wild-type currents.

Conclusions:

• Human population genomic data can effectively guide the selection of mutagenesis sites at ion channel interfaces.
• The K-KED interface residues play a significant role in regulating TRPV1 channel gating by stabilizing closed pore conformations.
• Combining structural, genomic, and functional data provides a powerful strategy for investigating ion channel molecular mechanisms.