Exocytotic insertion of TRPC6 channel into the plasma membrane upon Gq protein-coupled receptor activation.
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.
Cayouette
Sylvie
S
Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.
Lussier
Marc P
MP
Mathieu
Eve-Lyne
EL
Bousquet
Simon M
SM
Boulay
Guylain
G
D019921:Biotinylation; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis; D019084:Fluorescent Antibody Technique, Indirect; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; D013997:Time Factors; D014162:Transfection
D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D043562:Receptors, G-Protein-Coupled; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium