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.

Ca2+-permeable channels that are involved in the responses of mammalian cells to changes in extracellular osmolarity have not been characterized at the molecular level. Here we identify a new TRP (transient receptor potential)-like channel protein, OTRPC4, that is expressed at high levels in the kidney, liver and heart. OTRPC4 forms Ca2+-permeable, nonselective cation channels that exhibit spontaneous activity in isotonic media and are rapidly activated by decreases in, and are inhibited by increases in, extracellular osmolarity. Changes in osmolarity of as little as 10% result in significant changes in intracellular Ca2+ concentration. We propose that OTRPC4 is a candidate for a molecular sensor that confers osmosensitivity on mammalian cells.

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Proteins of the mammalian TRP (transient receptor potential) family form a heterogenous group of cation channels important for cellular Ca 2؉ signaling and homeostasis. Here we present the full-length sequence of TRPM3, a member of the melastatin-like subfamily (TRPM) of TRP channels. TRPM3 expression was found in human kidney and brain. HEK293 cells transiently transfected with TRPM3 showed a constitutive Ca 2؉ and Mn 2؉ entry. Whole-cell patch clamp experiments confirmed the spontaneous activity of TRPM3 and revealed permeability ratios P Ca /P Na of 1.57 and P Na /P Cs of 0.75. In cell-attached patches, spontaneous inward and outward currents were observed. At negative membrane potentials and in the presence of either 140 mM Cs ؉ , 140 mM Na ؉ , or 100 mM Ca 2؉ in the pipette solution, the single channel conductance levels were 133, 83, and 65 pS, respectively. The Ca 2؉ entry in TRPM3-expressing HEK293 cells increased during treatment with hypotonic extracellular solution. The reduction of extracellular osmolarity was accompanied by cell swelling, suggesting volume-regulated activity of TRPM3. From its function and expression in human kidney, we propose a role of TRPM3 in renal Ca 2؉ homeostasis.

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG͞ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein-cholesterol supercomplexes in the plasma membrane.prohibitin-domain proteins ͉ TRP channels ͉ DEG/ENaC channels ͉ slit diaphragm ͉ mechanosensation T he prohibitin homology (PHB)-domain proteins constitute a family of Ϸ1,800 proteins (SMART database; http:͞͞smart. embl-heidelberg.de) (1) all of which share an Ϸ150-aa domain similar to that in the mitochondrial protein prohibitin (2). More than 340 of these proteins, many of which have an N-terminal adjacent hydrophobic region that places them on the inner leaflet of the lipid bilayer, have been identified in animals. These membrane-associated proteins regulate osmotic homeostasis, mechanosensation, and cell signaling (3-5). Several of the animal PHB-domain proteins including flotillin, Podocin, prohibitin, stomatin, UNC-1, UNC-24, and the UNC-24-like mammalian protein SLP-1 are found in cholesterol-rich membrane fractions derived from the plasma membrane (reviewed in ref.2).In this article, we investigate the function of these proteins using two members of the family, MEC-2 from Caenorhabditis elegans and Podocin from mouse. MEC-2 (6) and Podocin (7) have a single, central hydrophobic domain that embeds these proteins in the inner leaflet of the plasma membrane with their N-and C-terminal tails facing the cytoplasm (Fig. 1a). Although the two proteins contain different N and C termini, they have hydrophobic regions that are 35% identical and 75% similar and PHB-domains that are 50% identical and 80% similar (Fig. 1b). The PHB domain is critical for the action of both proteins (8, 9).

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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.

The vanilloid receptor-related TRP channels (TRPV1-6) mediate thermosensation, pain perception and epithelial Ca2+ entry. As the specificity of TRPV channel heteromerization and determinants governing the assembly of TRPV subunits were largely elusive, we investigated the TRPV homo- and heteromultimerization. To analyze the assembly of TRPV subunits in living cells, we generated fluorescent fusion proteins or FLAG-tagged TRPV channel subunits. The interaction between TRPV subunits was assessed by analysis of the subcellular colocalization, fluorescence resonance energy transfer and coimmunoprecipitation. Our results demonstrate that TRPV channel subunits do not combine arbitrarily. With the exception of TRPV5 and TRPV6, TRPV channel subunits preferentially assemble into homomeric complexes. Truncation of TRPV1, expression of cytosolic termini of TRPV1 or TRPV4 and construction of chimeric TRPV channel subunits revealed that the specificity and the affinity of the subunit interaction is synergistically provided by interaction modules located in the transmembrane domains and in the cytosolic termini. The relative contribution of intramolecularly linked interaction modules presumably controls the overall affinity and the specificity of TRPV channel assembly.