Expression Level of the Canonical Transient Receptor Potential 3 (TRPC3) Channel Determines Its Mechanism of Activation

Abstract

Studies on the mechanism of activation of canonical transient receptor potential (TRPC) channels have often yielded conflicting results. In the current study, we have investigated the influence of expression level on the mode of regulation of TRPC3 channels. At relatively low levels of expression in DT40 chicken B-lymphocytes, TRPC3 was activated by the depletion of Ca 2؉ stores. Expression was increased by either transfecting with a 10-fold greater concentration of plasmid or transfecting with TRPC3 under control of a more efficient avian ␤-actin promoter. At higher levels of expression, TRPC3 was no longer store-operated but could be activated through receptor-coupled phospholipase C. Under these expression conditions, TRPC3 was efficiently activated in DT40 cells lacking inositol 1,4,5-trisphosphate receptors. The Ca 2؉ store-operated channels formed upon expression of TRPC3 at limited levels were blocked by gadolinium; the receptor-activated channels formed upon expression of higher levels of TRPC3 were insensitive to gadolinium. These findings indicate that a single ion channel protein can form or contribute to the formation of channels regulated in two very distinct ways, i.e. either by phospholipase C-derived messengers or Ca 2؉ store-depletion. The mechanism of regulation of the channels depends on their level of expression.In most nonexcitable cells, calcium signaling initiated through cell membrane receptors coupled to phospholipase C (PLC) 1 results in production of inositol 1,4,5-trisphosphate (IP 3 ) (1, 2). IP 3 induces the release of Ca 2ϩ from the endoplasmic reticulum and the subsequent influx of Ca 2ϩ across the plasma membrane through the capacitative calcium entry (CCE) or the store-operated calcium entry pathway (3-6). Although CCE has been widely studied in diverse cell types, the molecular identity of store-operated channels (SOCs) and the signal by which store emptying activates those channels remain uncertain. Mammalian homologues of the Drosophila transient receptor potential (TRP) channel have been proposed as candidates for SOCs (7,8). Among the members of the canonical TRP (TRPC) subfamily (designated TRPC1 through TRPC7), human TRPC3, first cloned by Zhu et al. (9), has been shown in many heterologous expression systems to behave as a receptor-activated channel with constitutive activity that cannot be further increased by Ca 2ϩ store depletion (10 -12). Furthermore, Hofmann et al. (13) showed that TRPC3 and its structural relative TRPC6 can be activated by diacylglycerols (DAGs), providing a possible mechanism of activation of these channels by phospholipase C-linked receptors independent of IP 3 and store depletion.We recently demonstrated that TRPC3 is regulated by store depletion when transiently expressed in DT40 chicken B-lymphocytes (14), and we proposed TRPC3 as a candidate for store-operated, non-selective cation channels. However, Venkatachalam et al. (15) reported that, in this same cell line, TRPC3 behaves as a receptor-activated channel with no dependence on the deple...

Chronic lithium treatment of B lymphoblasts from bipolar disorder patients reduces transient receptor potential channel 3 levels

“…While the lack of a correlation between the Li-induced decrements in the latter functional measure and TRPC3 immunolabeling relative to vehicle-treatment seems to conflict with this conclusion, it is not clear yet whether global TG-induced Ca 2 þ mobilization changes in direct proportion to the levels of TRPC3. The functional effects of TRPC3 in modulating SOCE have been shown to vary in relation to its expression levels: 23 at low levels TRPC3 operates as an SOCC, whereas at high expression levels it shows G protein receptor-coupled phospholipase C-operated characteristics. 21 The notion that pathophysiologically and diagnostically distinctive Ca 2 þ homeostasis abnormalities occur in BD-I disorder has grown out of series of observations of differential changes in indices of Ca 2 þ dynamics measured in surrogate peripheral blood cell and BLCLs models from BD, comparison mood and nonmood disorder patients, and healthy subjects.…”

“…9 Recent research directed at elaborating the molecular components and/or mechanisms mediating SOCE and ROCE highlights the key role of several members of the TRP family of cation permeable channels in these processes in nonexcitable cells, such as lymphocytes and platelets. [21][22][23] This superfamily of proteins is comprised of three branches distinguished by sequence homology and functional similarities: TRPC (canonical), TRPV (vanilloid) and TRPM (melastatin). 24 Two subgroups within the canonical branch, (TRPC1,4&5 and TRPC3,6&7, respectively) have been implicated in SOCE and ROCE, 24,25 raising the prospect of the involvement of one or more of these TRPC members in the Ca 2 þ abnormalities that have been reported in BD and the in actions of Li in BLCLs highlighted above.…”

Thapsigargin activates Ca2+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

“…However, numerous studies also demonstrated that TRPC channels did not operate as SOCE channels, but rather are gated by second messengers like Ca 2+ or diacylglycerol (DAG; [15][16][17]). Hence, the involvement of TRPC channels in the SOCE mechanism is a controversial issue, and appears to dependent on the cell types investigated, and importantly on the expression level of the TRPC [14,18,19].…”

Regulation of store-operated Ca2+entry by CD38 in human airway smooth muscle

“…In the last case, store-operated Ca 2ϩ entry (SOCE) is triggered by depletion of SR Ca 2ϩ stores (6,41,42,49,55,56). We and others have shown that different transient receptor potential channel (TRPC) isoforms are expressed in ASM (6,58).…”