Abstract
Phospholipase C signaling stimulates Ca2+ entry across the plasma membrane through multiple mechanisms. Ca2+ store depletion stimulates store-operated Ca2+-selective channels, or alternatively, other phospholipase C-dependent events activate Ca2+-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca2+ entry. No difference in the store-operated current I(crac) was detected between TRPC7-/- and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTPgammaS, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7-/- cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels.
Highlights
Phospholipase C signaling stimulates Ca2؉ entry across the plasma membrane through multiple mechanisms
Knock out of TRPC7 from DT40 B-lymphocytes produced three apparent phenotypes: an increased size of the Ca2ϩ stores, a diminished store-operated entry, and a diminished receptor- and diacylglycerolactivated entry. Only the latter, diacylglycerol-activated entry can be confidently attributed to the function of TRPC7 in native rapidly depleted with a combination of IP3 and calcium chelator, the development of the store-operated current Icrac was not significantly different in wild-type and TRPC7Ϫ/Ϫ cells
It has previously been argued that Transient receptor potential channels (TRPCs) channels can function in some environments as either storeoperated or non-store-operated channels [1], and when ectopically expressed in HEK293 cells, TRPC7 is clearly capable of functioning in both of these modes [6]
Summary
Phospholipase C signaling stimulates Ca2؉ entry across the plasma membrane through multiple mechanisms. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTP␥S, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7؊/؊ cells but could be restored by transfection with human TRPC7. In B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors This represents the first demonstration of a physiological function for endogenous TRPC7 channels. The calcium-permeable cation channel, canonical transient receptor potential 7 (TRPC7) was originally shown to function as a phospholipase C-regulated channel, presumably through production of diacylglycerol [4]. In the avian B-cell line, TRPC7 appears to function as a phospholipase C-regulated, diacylglycerol-activated channel
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