Molecular cloning and functional characterization of a novel receptor- activated TRP Ca²⁺ channel from mouse brain

Characterization of mammalian homologues of Drosophila TRP proteins, which induce light-activated Ca²⁺ conductance in photoreceptors, has been an important clue to understand molecular mechanisms underlying receptor- activated Ca²⁺ influx in vertebrate cells. We have here isolated cDNA that encodes a novel TRP homologue, TRP5, predominantly expressed in the brain. Recombinant expression of the TRP5 cDNA in human embryonic kidney cells dramatically potentiated extracellular Ca²⁺-dependent rises of intracellular Ca²⁺ concentration ([Ca²⁺](i)) evoked by ATP. These [Ca²⁺](i) transients were inhibited by SKandF96365, a blocker of receptor- activated Ca²⁺ entry, and by La³⁺. Expression of the TRP5 cDNA, however, did not significantly affect [Ca²⁺](i) transients induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺-ATPases. ATP stimulation of TRP5-transfected cells pretreated with thapsigargin to deplete internal Ca²⁺ stores caused intact extracellular Ca²⁺-dependent [Ca²⁺](i) transients, whereas ATP suppressed [Ca²⁺](i) in thapsigargin-pretreated control cells. Furthermore, in ATP-stimulated, TRP5-expressing cells, there was no significant correlation between Ca²⁺ release from the internal Ca²⁺ store and influx of extracellular Ca²⁺. Whole-cell mode of patchclamp recording from TRP5-expressing cells demonstrated that ATP application induced a large inward current in the presence of extracellular Ca²⁺. Omission of Ca²⁺ from intrapipette solution abolished the current in TRP5-expressing cells, whereas 10 nM intrapipette Ca²⁺ was sufficient to support TRP5 activity triggered by ATP receptor stimulation. Permeability ratios estimated from the zero-current potentials of this current were P(Ca):P(Na):P(Cs) = 14.3:1.5:1. Our findings suggest that TRP5 directs the formation of a Ca²⁺-selective ion channel activated by receptor stimulation through a pathway that involves Ca²⁺ but not depletion of Ca²⁺ store in mammalian cells.