Abstract
Mammalian transient receptor potential channels (TRPCs) form a family of Ca(2+)-permeable cation channels currently consisting of seven members, TRPC1-TRPC7. These channels have been proposed to be molecular correlates for capacitative Ca(2+) entry channels. There are only a few studies on the regulation and properties of the subfamily consisting of TRPC4 and TRPC5, and there are contradictory reports concerning the possible role of intracellular Ca(2+) store depletion in channel activation. We therefore investigated the regulatory and biophysical properties of murine TRPC4 and TRPC5 (mTRPC4/5) heterologously expressed in human embryonic kidney cells. Activation of G(q/11)-coupled receptors or receptor tyrosine kinases induced Mn(2+) entry in fura-2-loaded mTRPC4/5-expressing cells. Accordingly, in whole-cell recordings, stimulation of G(q/11)-coupled receptors evoked large, nonselective cation currents, an effect mimicked by infusion of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). However, depletion of intracellular Ca(2+) stores failed to activate mTRPC4/5. In inside-out patches, single channels with conductances of 42 and 66 picosiemens at -60 mV for mTRPC4 and mTRPC5, respectively, were stimulated by GTPgammaS in a membrane-confined manner. Thus, mTRPC4 and mTRPC5 form nonselective cation channels that integrate signaling pathways from G-protein-coupled receptors and receptor tyrosine kinases independently of store depletion. Furthermore, the biophysical properties of mTRPC4/5 are inconsistent with those of I(CRAC), the most extensively characterized store-operated current.
Highlights
Mammalian homologues of the Drosophila cation channel TRP form a novel gene family within the superfamily of cation channels with six transmembrane segments (1)
Subcellular Localization of mTRPC4 and mTRPC5 Fused to Green Fluorescent Protein—To visualize the subcellular localization of the presumptive cation channels in living human embryonic kidney (HEK) cells, cDNA constructs encoding mTRPC4, mTRPC5, and hTRPC6 C-terminally fused to Enhanced green fluorescent protein (eGFP) were generated
The most prominent mTRPC4-eGFP and hTRPC6-eGFP signals were found in the plasma membrane, whereas a considerable amount of mTRPC5-eGFP was retained in a perinuclear compartment, probably the Golgi apparatus (Fig. 1E)
Summary
Mammalian homologues of the Drosophila cation channel TRP form a novel gene family within the superfamily of cation channels with six transmembrane segments (1). Activation of PLCs by extracellular signaling molecules leads to the production of inositol 1,4,5trisphosphate (InsP3) and diacylglycerol and couples to intracellular signaling cascades by increasing the cytosolic Ca2ϩ concentration ([Ca2ϩ]i). These changes in [Ca2ϩ]i result from InsP3-mediated Ca2ϩ release from intracellular stores and/or Ca2ϩ entry from the extracellular space. From the close structural relationship of TRPC4 and TRPC5, similar regulatory and biophysical properties might be expected Both bovine TRPC4 and rabbit TRPC5 have been reported to form store-operated, relatively Ca2ϩ-selective channels with an inwardly rectifying current-voltage relation (9, 10). The available data on TRPC4/5 cannot be integrated into a consistent model of signaling pathways leading to activation of these channels
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