Exocytotic Insertion of TRPC6 Channel into the Plasma Membrane upon Gq Protein-coupled Receptor Activation

Sylvie Cayouette,Marc P Lussier,Eve-Lyne Mathieu,Simon M Bousquet,Guylain Boulay

doi:10.1074/jbc.m312042200

Abstract

TRPC proteins are the mammalian homologues of the Drosophila transient receptor potential channel and are involved in calcium entry after agonist stimulation of non-excitable cells. Seven mammalian TRPCs have been cloned, and their mechanisms of activation and regulation are still the subject of intense research. TRPC proteins interact with the inositol 1,4,5-trisphosphate receptor, and the conformational coupling plays a critical role in the activation of calcium entry. Some evidence also supports an exocytotic mechanism as part of the activation of calcium entry. To investigate the possible involvement of exocytosis in TRPC6 activation, we evaluated the location of TRPC6 at the plasma membrane by biotinylation labeling of cell surface proteins and by indirect immunofluorescence marking of TRPC6 in stably transfected HEK 293 cells. We showed that when the muscarinic receptor was stimulated or the thapsigargin-induced intracellular calcium pool was depleted the level of TRPC6 at the plasma membrane increased. The carbachol concentration at which TRPC6 externalization occurred was lower than the concentration required to activate TRPC6. Externalization occurred within the first 30 s of stimulation, and TRPC6 remained at the plasma membrane as long as the stimulus was present. These results indicate that an exocytotic mechanism is involved in the activation of TRPC6.

Highlights

Increases in intracellular calcium ([Ca2ϩ]i) regulate important cellular functions, including cell growth, differentiation, contraction, and secretion [1, 2]
We used a protocol that relies on caveolae-related microdomains (CRM) resistance to solubilization in Triton X-100 at low temperatures and high buoyancy in sucrose density gradients to assess the presence of TRPC6 in CRMs
Stimulation of T6.11 HEK cells with CCh, an agonist of the endogenous muscarinic receptor, or thapsigargin, a sarco(endo)plasmic reticulum calcium ATPase pump inhibitor known to stimulate Ca2ϩ entry, did not increase the amount of TRPC6 or IP3 receptor in this fraction. These results suggest that a stable complex between TRPC6 and IP3 receptor is already formed under basal conditions and is not enhanced by stimulation of the cells

Summary

Introduction

Increases in intracellular calcium ([Ca2ϩ]i) regulate important cellular functions, including cell growth, differentiation, contraction, and secretion [1, 2]. We showed that when the muscarinic receptor was stimulated or the thapsigargin-induced intracellular calcium pool was depleted the level of TRPC6 at the plasma membrane increased. Overexpression studies showed that the homotetramer channel consisting of TRPC1, TRPC4, or TRPC5 can be activated either by the depletion of the intracellular Ca2ϩ pool (8 –10) or by the activation of Gq protein-coupled receptor stimulation [11, 12].

Results

Conclusion