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.Increases in intracellular calcium ([Ca 2ϩ ] i ) regulate important cellular functions, including cell growth, differentiation, contraction, and secretion (1, 2). The increase in [Ca 2ϩ ] i is initiated by the activation of phospholipase C or -␥ by a G q protein-coupled receptor or tyrosine kinase receptor, respectively, causing the hydrolysis of phosphatidylinositol 4,5-bisphosphate and generating two-second messengers, inositol 1,4,5-trisphosphate (IP 3 ) 1 and diacylglycerol. IP 3 binds to its specific receptor on the endoplasmic reticulum and causes the first phase of Ca 2ϩ mobilization by releasing Ca 2ϩ from the intracellular pool. The second phase involves Ca 2ϩ entry through the plasma membrane, which maintains [Ca 2ϩ ] i above basal levels. No cellular activity has ever been observed in the absence of this second phase of Ca 2ϩ mobilization.The TRPC protein family is involved in Ca 2ϩ entry (3-5). Seven genes have been identified in the mammalian genome by homology with Drosophila TRP and TRPL, two proteins that are responsible for the light-induced current in insect phototransduction. TRPC channels are probably composed of four subunits, each containing three to four ankyrin-like repeats, two predicted coiled-coil regions (one in the amino terminus and another one in the carboxyl terminus), and a membranespanning region. The amino acids that link the fifth and sixth transmembrane segments form the putative pore region loop. Recent studies on the subunit arrangement of native TRPC in rat brain synaptosomes and the overexpression of TRPC in various systems suggest that the TRPC protein family can be subdivided in two distinct groups and that...