MOLECULAR-CLONING AND FUNCTIONAL-CHARACTERIZATION OF A NOVEL RECEPTOR-ACTIVATED TRP CA2-BRAIN( CHANNEL FROM MOUSE)

Characterization of mammalian homologues of Drosophila TRP proteins, w hich induce light-activated Ca2+ conductance in photoreceptors, has be en an important clue to understand molecular mechanisms underlying rec eptor-activated Ca2+ influx in vertebrate cells. We have here isolated cDNA that encodes a novel TRP homologue, TRP5, predominantly expresse d in the brain. Recombinant expression of the TRP5 cDNA in human embry onic kidney cells dramatically potentiated extracellular Ca2+-dependen t rises of intracellular Ca2+ concentration ([Ca2+](i)) evoked by ATP. These [Ca2+](i) transients were inhibited by SK&F96365, a blocker of receptor-activated Ca2+ entry, and by La3+, Expression of the TRP5 cDN A, however, did not significantly affect [Ca2+](i) transients induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPases. A TP stimulation of TRP5-transfected cells pretreated with thapsigargin to deplete internal Ca2+ stores caused intact extracellular Ca2+-depen dent [Ca2+](i) transients, whereas ATP suppressed [Ca2+](i) in thapsig argin-pretreated control cells. Furthermore, in ATP-stimulated, TRP5-e xpressing cells, there was no significant correlation between Ca2+ rel ease from the internal Ca2+ store and influx of extracellular Ca2+. Wh ole-cell mode of patch-clamp recording hom TRP5-expressing cells demon strated that ATP application induced a large inward current in the pre sence of extracellular Ca2+. Omission of Ca2+ from intrapipette soluti on abolished the current in TRP5-expressing cells, whereas 10 nM intra pipette Ca2+ was sufficient to support TRP5 activity triggered by ATP receptor stimulation. Permeability ratios estimated from the zero-curr ent potentials of this current were P-Ca:P-Na:P-Cs = 14.3:1.5:1, Our f indings suggest that TRP5 directs the formation of a Ca2+-selective io n channel activated by receptor stimulation through a pathway that inv olves Ca2+ but not depletion of Ca2+ store in mammalian cells.