Native TRPC7 Channel Activation by an Inositol Trisphosphate Receptor-dependent Mechanism

Abstract

In DT40 B lymphocytes, Canonical Transient Receptor Potential 7 (TRPC7) functions as a diacylglycerol-activated non-selective cation channel. However, previous work indicated that the non-store-operated Ca 2؉ entry in this cell type depends upon inositol trisphosphate receptors (IP 3 R). With the cell-attached configuration oleyl-acetyl-glycerol (OAG) induced single channel activity (75 pS) that was not observed in TRPC7 ؊/؊ DT40 cells were indistinguishable from one another and from wildtype cells. Thus, TRPC7 forms, or is part of, the channel underlying endogenous diacylglycerol-activated currents in DT40 B lymphocytes, and this activity of native TRPC7 requires IP 3 R. However, with conditions expected to produce greater expression levels, TRPC7 functioned independently of the presence of IP 3 R. This finding may serve to resolve previously conflicting reports from expression studies of TRPC channels. Canonical Transient Receptor Potential (TRPC)2 channels, which belong to the larger superfamily of mammalian TRP channel-forming proteins, are among the most important neurotransmitter and hormone-regulated cation channels in nonexcitable cells (1, 2). Physiologically, TRPC channels function as multifunctional calcium-permeable cation channels that can be activated through the phospholipase C (PLC) pathway. However, whether individual TRPC members function as store-operated Ca 2ϩ channels (whose activation is initiated by depletion of Ca 2ϩ stores) and/or non-store-operated Ca 2ϩ channels following PLC activation is still a matter of debate. Some of the confusion stems from the fact that, when heterologously expressed, their mode of regulation seems to be strongly dependent on cell type, expression level, or expression environment (discussed in Ref.3). TRPC7 for instance, a member of the TRPC3/6/7 subfamily, was originally shown to function as a PLC-regulated channel (4), presumably through PLC-derived diacylglycerol (DAG), but subsequent evidence indicated that it could also function as a store-operated Ca 2ϩ channel (5). Recent work from our laboratory demonstrated that these apparently contradictory findings likely resulted from differences in expression conditions (6). In an attempt to determine which of these two behaviors may correspond to the physiological function of native, endogenously expressed TRPC7, we recently utilized targeted homologous recombination to knock out TRPC7 in DT40 B lymphocytes. We found that knock out of TRPC7 did not significantly affect the store-operated calciumselective current I crac known to be expressed in these cells (7). Rather, in the avian B-cell line, TRPC7 appears to function as a PLC-regulated, DAG-activated non-selective cation channel. However, previous studies from this laboratory (8) and others (9, 10) showed that in DT40 cells non-store-operated Ca 2ϩ , receptor-regulated cation entry depends in some manner on inositol trisphosphate (IP 3 ) receptors, but the precise role of IP 3 receptors (IP 3 R) in this pathway has not been defined. Because a Ca 2ϩ -impermeable...

TRPC channels as STIM1-regulated store-operated channels

“…When it is expressed at high levels, TRPC7 functions as a storeindependent channel that is inhibited by high concentration of Gd 3+ . By contrast, at low expression level TRPC7 behaves as a SOC, its activity depends on the presence of IP 3 receptors and it is highly sensitive to inhibition by Gd 3+ [25][26][27]. Hence, the function of TRPC7 as SOCs depends on its expression level.…”

Complex regulation of the TRPC3, 6 and 7 channel subfamily by diacylglycerol and phosphatidylinositol-4,5-bisphosphate

“…Therefore statistical evaluations of effects were based on paired t-tests. Detailed methods for collection and analysis of TRPC7 single channel data were described previously [14].…”

“…Therefore statistical evaluations of effects were based on paired t-tests. Detailed methods for collection and analysis of TRPC7 single channel data were described previously [14].All reagents were purchased from Sigma (St. Louis, MO), except oleyl acetyl glycerol (OAG) (Calbiochem, San Diego, CA) and PIP 2 (Echelon Biosciences Inc., Salt Lake City, UT). …”

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].…”