Eukaryotic cells respond to many hormones and neurotransmitters with increased activity of the enzyme phospholipase C and a subsequent rise in the concentration of intracellular free calcium ([Ca2+]i). The increase in [Ca2+]i occurs as a result of the release of Ca2+ from intracellular stores and an influx of Ca2+ through the plasma membrane; this influx of Ca2+ may or may not be store-dependent. Drosophila transient receptor potential (TRP) proteins and some mammalian homologues (TRPC proteins) are thought to mediate capacitative Ca2+ entry. Here we describe the molecular mechanism of store-depletion-independent activation of a subfamily of mammalian TRPC channels. We find that hTRPC6 is a non-selective cation channel that is activated by diacylglycerol in a membrane-delimited fashion, independently of protein kinases C activated by diacylglycerol. Although hTRPC3, the closest structural relative of hTRPC6, is activated in the same way, TRPCs 1, 4 and 5 and the vanilloid receptor subtype 1 are unresponsive to the lipid mediator. Thus, hTRPC3 and hTRPC6 represent the first members of a new functional family of second-messenger-operated cation channels, which are activated by diacylglycerol.

Mammalian transient receptor potential channels (TRPCs) form a family of Ca2؉ -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 (GTP␥S). However, depletion of intracellular Ca2؉ 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 GTP␥S 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.

Depletion of intracellular calcium stores generates a signal that activates Ca2+-permeable channels in the plasma membrane. We have identified a human cDNA, TRPC1A, from a human fetal brain cDNA library. TRPC1A is homologous to the cation channels trp and trpl in Drosophila and is a splice variant of the recently identified cDNA Htrp-1. Expression of TRPC1A in CHO cells induced nonselective cation currents with similar permeabilities for Na+, Ca2+, and Cs+. The currents were activated by intracellular infusion of myo inositol 1,4,5-trisphosphate or thapsigargin. Expression of TRPC1A significantly enhanced increases in the intracellular free calcium concentration induced by Ca2+ restitution after prolonged depletion. Similar results were obtained in Sf9 cells. We conclude that TRPC1A encodes a Ca2+-permeable cation channel activated by depletion of intracellular calcium stores.

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.

TRPC6-dependent Ca2+ flux in endothelial cells after TLR4-induced diacylglycerol production mediates lung endothelial barrier disruption and inflammation induced by LPS.