Activation of Human TRPC6 Channels by Receptor Stimulation

Abstract

The human TRPC6 channel was expressed in human embryonic kidney (HEK) cells, and activity was monitored using the giga-seal technique. Whole cell membrane currents with distinctive inward and outward rectification were activated by carbachol (CCh) in TRPC6-expressing cells, but not in lacZ-transfected controls. The effect of CCh was steeply dose-dependent with a K 0.5 of ϳ10 M and a Hill coefficient of 3-4. A steep concentration-response relationship was also observed when TRPC6 activity was measured using a fluorescence-based imaging plate reader (FLIPR) assay for membrane depolarization. Ionomycin, thapsigargin, and dialysis of the cell with inositol 1,4,5-trisphosphate via the patch pipette had no effect on TRPC6 currents, but exogenous application of 1-oleoyl acetyl-sn-glycerol (OAG, 30 -300 M) produced a slow increase in channel activity. The PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.5 M) had no significant acute effect on TRPC6, or on the subsequent response to OAG. In contrast, the response to CCh was blocked >90% by PMA pretreatment. To further explore the role of DAG in receptor stimulation, TRPC6 currents were monitored following the sequential addition of CCh and OAG. Surprisingly, concentrations of CCh that produced little or no response in the absence of OAG, produced increases in TRPC6 currents in the presence of OAG that were larger than the sum of either agent alone. Likewise, the response to OAG was superadditive following prior stimulation of the cells with near threshold concentrations of CCh. Overall, these results suggest that generation of DAG alone may not fully account for activation of TRPC6, and that other receptor-mediated events act synergistically with DAG to stimulate channel activity. This synergy may explain, at least in part, the steep dose-response relationship observed for CCh-induced TRPC6 currents expressed in HEK cells.

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

“…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).…”

“…RGS proteins are GTPase-activating proteins that enhance G␣-GTP hydrolysis, thereby decreasing the lifetime of active states of G protein subunits, and RGS4 is expressed in ␤ cells (22). TRPC6 has been reported to be a receptor-operated cation channel that is activated upon GPCR stimulation and subsequent phospholipase C ␤ activation (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).…”

Role of TRPC3 channels in ATP-induced Ca2+signaling in principal cells of the inner medullary collecting duct

“…Since the actual mechanism of activation of TRPC3 channels by DAG remains unknown, the reason for this difference is not clear. We and others also have shown that activation of TRPC6 by exogenous DAG is less than that observed with receptor stimulation (6,13), and it has been suggested that DAG may act indirectly to activate TRPC3 channels (31). Although it seems clear that exogenous DAG is not equivalent to receptor-mediated activation, there is good correlation between the extent of TRPC3 channel activation and the subsequent transepithelial Ca 2ϩ flux in the IMCD-3 cell model system.…”

“…There are seven members of the mammalian TRPC family, designated TRPC1-TRPC7, all of which are activated downstream of PLC (22). Although the ability of TRPC proteins to function as native store-operated channels remains highly controversial (5,24,35), it is well established that TRPC3, TRPC6, and TRPC7 can be activated by DAG analogs (6,10,23,24,33), but the molecular basis for this activation remains unknown.Our previous studies have shown that TRPC3 and TRPC6 are expressed in principal cells of the collecting duct (CD) along with the water channel, aquaporin-2 (AQP2) (7,8). When examined in normally hydrated rats, all three channels are predominantly localized to cytoplasmic vesicles.…”

The Mechanosensitive Ca2+ Channel as a Central Regular of Prostate Tumor Cell Migration and Invasiveness

“…The general consensus is that TRPC6 forms a ROC that is directly activated by DAG, and is insensitive to activation by IP 3 and Ca 2+ store depletion (Boulay et al 1997;Estacion et al 2004;Zagranichnaya et al 2005;Zhang et al 2006). Although TRPC6 is a member of the TRPC3/6/7 subfamily it shows distinct functional and structural properties.…”