A TRPV4 Channel C-terminal Folding Recognition Domain Critical for Trafficking and Function

Lei Lei,Xu Cao,Fan Yang,Di-Jing Shi,Yi-Quan Tang,Jie Zheng,Kewei Wang

doi:10.1074/jbc.m113.457291

Abstract

The Ca(2+)-permeable transient receptor potential vanilloid subtype 4 (TRPV4) channel mediates crucial physiological functions, such as calcium signaling, temperature sensing, and maintaining cell volume and energy homeostasis. Noticeably, most disease-causing genetic mutations are concentrated in the cytoplasmic domains. In the present study, we focused on the role of the TRPV4 C terminus in modulating protein folding, trafficking, and activity. By examining a series of C-terminal deletions, we identified a 20-amino acid distal region covering residues 838-857 that is critical for channel folding, maturation, and trafficking. Surface biotinylation, confocal imaging, and fluorescence-based calcium influx assay demonstrated that mutant proteins missing this region were trapped in the endoplasmic reticulum and unglycosylated, leading to accelerated degradation and loss of channel activity. Rosetta de novo structural modeling indicated that residues 838-857 assume a defined conformation, with Gly(849) and Pro(851) located at critical positions. Patch clamp recordings confirmed that lowering the temperature from 37 to 30 °C rescued channel activity of folding-defective mutants. Moreover, biochemical tests demonstrated that, in addition to participating in C-C interaction, the C terminus also interacts with the N terminus. Taken together, our findings indicate that the C-terminal region of TRPV4 is critical for channel protein folding and maturation, and the short distal segment plays an essential role in this process. Therefore, selectively disrupting the folding-sensitive region may present therapeutic potential for treating overactive TRPV4-mediated diseases, such as pain and skeletal dysplasias.

Highlights

Overactive transient receptor potential vanilloid subtype 4 (TRPV4) mutants cause human diseases, such as skeletal dysplasias
Our objective was to elucidate the role of the TRPV4 C terminus in regulating channel activity because mutations in intracellular regions are known to cause human diseases as a result of altered channel activity
Using a combination of biochemical approaches, confocal imaging, calcium influx assay, and electrophysiology, we identified a novel narrow region in the C terminus of TRPV4 whose disruption ablates channel activity

Summary

Introduction

Overactive TRPV4 mutants cause human diseases, such as skeletal dysplasias. Significance: Targeting the folding recognition domain presents attractive therapeutic potential for overactive TRPV4-mediated pathology. By examining a series of C-terminal deletions, we identified a 20-amino acid distal region covering residues 838 – 857 that is critical for channel folding, maturation, and trafficking. Confocal imaging, and fluorescence-based calcium influx assay demonstrated that mutant proteins missing this region were trapped in the endoplasmic reticulum and unglycosylated, leading to accelerated degradation and loss of channel activity. Our findings indicate that the C-terminal region of TRPV4 is critical for channel protein folding and maturation, and the short distal segment plays an essential role in this process. Selectively disrupting the folding-sensitive region may present therapeutic potential for treating overactive TRPV4-mediated diseases, such as pain and skeletal dysplasias

Objectives

Results

Conclusion