Regulation of Plasma Membrane Expression of TRPV4 Channel by GSK1016790A and Hypotonic Stress in HeLa and M‐1 Cells

Abstract

TRPV4 (Transient Receptor Potential Vanilloid 4) channels are activated by a wide range of stimuli, including hypotonic stress, phorbol ester 4α‐PDD, non‐noxious heat and mechanical stress. GSK1016790A (GSK101, GlaxoSmithKline), a potent and specific activator of TRPV4 was evaluated in HeLa cells transiently transfected with TRPV4 (HeLa‐TRPV4) and M‐1 (mouse cortical collecting duct) cells expressing TRPV4 endogenously. GSK101 (10 nM) causes a TRPV4 specific calcium influx in both over‐expressed and endogenous cell models for TRPV4, which can be inhibited by Ruthenium Red (RR). Hypotonic stress (225 mOsm/kg) causes a calcium influx in HeLa‐TRPV4 and M‐1 cells, but only partially responds to RR inhibition. Plasma membrane proteins were extracted and Western blotting was utilized to study the expression of TRPV4 over a time course of 30 minutes under GSK101 and hypotonic stress treatment. GSK101 causes a steady down‐regulation of TRPV4 plasma membrane expression over 30 minutes, while the hypotonic stress causes a more complex change in TRPV4 plasma membrane expression. Fluorescence confocal microscopy shows co‐localization of TRPV4 with microtubules and actin, suggesting likely trafficking and endocytosis of TRPV4 channel. Immunocytochemistry also shows co‐localization of TRPV4 with large‐conductance calcium‐activated potassium channels (BK), indicating a potential macro‐complex of these channels are probably involved in intracellular calcium and volume regulation. It is concluded that GSK101 specifically activates TRPV4 in HeLa‐TRPV4 cells and M‐1 cells, which likely involves regulation of endocytosis of TRPV4 from the plasma membrane. Hypotonic stress activates TRPV4 in a more complex pattern of regulation that may involve recycling.Supported by NIH DK70950.