Mammalian homologues of the Drosophila transient receptor potential (TRP) channel gene encode a family of at least 20 ion channel proteins. They are widely distributed in mammalian tissues, but their specific physiological functions are largely unknown. A common theme that links the TRP channels is their activation or modulation by phosphatidylinositol signal transduction pathways. The channel subunits have six transmembrane domains that most probably assemble into tetramers to form non-selective cationic channels, which allow for the influx of calcium ions into cells. Three subgroups comprise the TRP channel family; the best understood of these mediates responses to painful stimuli. Other proposed functions include repletion of intracellular calcium stores, receptor-mediated excitation and modulation of the cell cycle.

TRP proteins are cation channels responding to receptor-dependent activation of phospholipase C. Mammalian (TRPC) channels can form hetero-oligomeric channels in vitro, but native TRPC channel complexes have not been identified to date. We demonstrate here that TRPC1 and TRPC5 are subunits of a heteromeric neuronal channel. Both TRPC proteins have overlapping distributions in the hippocampus. Coexpression of TRPC1 and TRPC5 in HEK293 cells resulted in a novel nonselective cation channel with a voltage dependence similar to NMDA receptor channels, but unlike that of any reported TRPC channel. TRPC1/TRPC5 heteromers were activated by G(q)-coupled receptors but not by depletion of intracellular Ca(2+) stores. In contrast to the more common view of the TRP family as comprising store-operated channels, we propose that many TRPC heteromers form diverse receptor-regulated nonselective cation channels in the mammalian brain.

Mammalian short TRP channels (TRPCs) are putative receptor-and store-operated cation channels that play a fundamental role in the regulation of cellular Ca 2؉ homeostasis. Assembly of the seven TRPC homologs (TRPC1-7) into homo-and heteromers can create a large variety of different channels. However, the compositions as well as the functional properties of native TRPC complexes are largely undefined. We performed a systematic biochemical study of TRPC interactions in mammalian brain and identified previously unrecognized channel heteromers composed of TRPC1, TRPC4, or TRPC5 and the diacylglycerol-activated TRPC3 or TRPC6 subunits. The novel TRPC heteromers were found exclusively in embryonic brain. In heterologous systems, we demonstrated that assembly of these novel heteromers required the combination of TRPC1 plus TRPC4 or TRPC5 subunits along with diacylglycerolsensitive subunits in the channel complexes. Functional interaction of the TRPC subunits was verified using a dominant negative TRPC5 mutant (TRPC5 DN ). Co-expression of TRPC5 DN suppressed currents through TRPC5-and TRPC4-containing complexes; TRPC3-associated currents were unaffected by TRPC5 DN unless TRPC1 was also co-expressed. This complex assembly mechanism increases the diversity of TRPC channels in mammalian brain and may generate novel heteromers that have specific roles in the developing brain.Cellular Ca 2ϩ signaling is dependent on ubiquitously expressed receptor-and store-operated cation channels (ROCs 1 and SOCs). These channels mediate Ca 2ϩ influx in response to hormones and other stimuli that activate phospholipase C (PLC) isoenzymes (1-3). The widespread expression and functional diversity of ROCs and SOCs is reflected by the large variety of these channels with diverse biophysical properties and regulation mechanisms.There is growing evidence that members of the TRPC cation channel family can form ROCs and SOCs. Belonging to the larger superfamily of mammalian TRP channels, seven TRPCs (TRPC1-7) have been identified (4, 5). Heterologous expression of the highly homologous TRPC3, 6, and 7 gave rise to diacylglycerol (DAG)-activated ROCs (6, 7). TRPC1, 4, and 5 constitute a second structural TRPC subfamily. Whereas TRPC4 and TRPC5 were activated by an unknown PLC-dependent mechanism (8, 9), TRPC1 was characterized as a SOC (10). Other studies indicated that TRPC3, TRPC4, and TRPC5 might also contribute to SOCs activated by depletion of intracellular Ca 2ϩ stores (11)(12)(13)(14).Based on their structural similarity with voltage-dependent K ϩ channels, functional TRPC complexes are presumed to be tetramers. Many of the disparate results regarding TRPC function and regulation could be reconciled by assuming that TRPC subunits can assemble into heteromeric channels with diverse properties. Montell and co-workers demonstrated that TRPC1 and TPRC3 formed complexes based on co-immunoprecipitation studies of tagged proteins (15). We reported that TRPC1 co-assembled with TRPC4 and TRPC5 in rat brain (16). The biophysical properties of TRPC(1...

Embryonic stem (ES) cells represent a suitable model to analyze cell differentiation processes in vitro. Here, we report that pluripotent ES cells of the line BLC 6 differentiate in vitro into neuronal cells possessing the complex electrophysiological and immunocytochemical properties of postmitotic nerve cells. In the course of differentiation BLC 6-derived neurons differentially express voltage-dependent (K+, Na+, Ca2+) and receptor-operated (GABAA, glycine, AMPA, NMDA receptors) ionic channels. They generate fast Na(+)-driven action potentials and are functionally coupled by inhibitory (GABAergic) and excitatory (glutamatergic) synapses as revealed by measurements of postsynaptic currents. Moreover, BLC 6-derived neurons express neuron-specific cytoskeletal, cell adhesion and synaptic vesicle proteins and exhibit a Ca(2+)-dependent GABA secretion. Thus, the ES cell model enables the investigation of cell lineage determination and signaling mechanisms in the developing nervous system from a pluripotential stem cell to a differentiated postmitotic neuron. The in vitro differentiation of neurons from ES cells may be an excellent approach to study by targeted gene disruption a variety of neuronal functions.