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

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.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...

Muscarinic Control of MIN6 Pancreatic β Cells Is Enhanced by Impaired Amino Acid Signaling

“…We also examined TRPC6 because a microarray analysis suggested that it was up-regulated in T1R3 knockdown cells. Carbachol is capable of inducing ROCE by activating and promoting cell surface expression of TRPC6 downstream of muscarinic receptor binding (26,27). Although the suspected change in its mRNA could not be validated, TRPC6 seemed a logical candidate and may be involved in some manner we did not detect (23,24).…”

“…Calcium entry occurring through plasma membrane channels as a result of GPCR activation independent of the state of Ca 2ϩ i stores is referred to as ROCE (25). It has been demonstrated that carbachol is capable of inducing ROCE by activating and promoting cell surface expression of TRPC6 downstream of muscarinic receptor binding (26,27). Expression of neither RGS4 nor TRPC6 was significantly different in T1R3 knockdown cells compared with the control, suggesting that the enhanced carbachol response was not a result of decreased RGS4 or increased TRPC6 expression (Fig.…”

Arachidonic Acid–Induced Ca2+ Entry Is Involved in Early Steps of Tumor Angiogenesis

“…The presence of a channel at the cell membrane must be tightly controlled to ensure correct timing and duration of signals relayed by such proteins. Activation of several channels belonging to the TRP family of channels correlates with their increased appearance at the plasma membrane (53)(54)(55). The molecular nature of AA-activated, calcium-permeable channels in the plasma membrane is still unknown.…”

Homer proteins in Ca2+ signaling by excitable and non-excitable cells

“…Another study reported translocation of TRPC3 in response to stimulation of the EGF receptor but failed to observe translocation of TRPC3 in response to M3 receptor stimulation [50]. Stimulation of the M3 receptor causes the translocation of TRPC6 to the plasma membrane [51], and stimulation EGF receptor caused the translocation of TRPC4 [52] and TRPC5 [53] to the plasma membrane. The PI3-kinase pathway and its downstream effector Rac1 are implicated in the EGFstimulated translocation of TRPC5 [53], which may be a general mechanism mediating Tyrosine kinase receptor mediated translocation of TRPC channels.…”