Profiling the TRPV4 ankyrin repeat domain interactome and its disruption by neuromuscular disease-causing mutations

Area of Science:

• Ion channel biophysics
• Molecular cell biology
• Genetics of neuromuscular disorders

Background:

• Ankyrin repeat domains (ARDs) are crucial protein-protein interaction motifs found in eukaryotic proteins.
• In the transient receptor potential (TRP) superfamily, ARDs are located in the N-termini of ion channels, including TRPV4.
• Gain-of-function mutations in TRPV4 associated with neuromuscular diseases cluster on the ARD surface, suggesting its critical role.

Purpose of the Study:

• To comprehensively profile the interactome of the wild-type (WT) and mutant TRPV4 ARD.
• To identify how disease-causing mutations alter the TRPV4 ARD interactome.
• To investigate the functional consequences of altered interactions on TRPV4 channel activity and neuromuscular disease pathogenesis.

Main Methods:

• Human proteome microarray analysis to identify TRPV4 ARD interactors.
• Co-immunoprecipitation assays to validate protein interactions in cell-based models.
• Calcium imaging studies to assess TRPV4 channel activity in response to genetic manipulations.

Main Results:

• The TRPV4 WT-ARD interacts with 78 proteins, including NEDD4L and ARHGEF10, involved in ubiquitination and small GTPase signaling.
• Twenty-one TRPV4 ARD interactions are significantly affected by disease-causing mutations.
• ARHGEF10, also mutated in neuromuscular disease, shows reduced binding to mutant TRPV4 and its inhibitory effect on TRPV4 activity is lost in the presence of mutations.

Conclusions:

• The TRPV4 ARD is a hub for diverse protein interactions critical for channel function.
• Disease-associated mutations disrupt specific TRPV4 ARD interactions, leading to altered channel activity.
• Dysregulation of ARHGEF10-TRPV4 interactions may contribute to the pathophysiology of neuromuscular diseases.