EXPRESSION OF DROSOPHILA TRPL CRNA IN XENOPUS-LAEVIS OOCYTES LEADS TOTHE APPEARANCE OF A CA2-THIO]TRIPHOSPHATE( CHANNEL ACTIVATED BY CA2+ AND CALMODULIN, AND BY GUANOSINE 5'[GAMMA)

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull an d Kelly (1992) Neuron 8, 631-642], on divalent cation inflow in Xenopu s laevis oocytes were investigated. The addition of extracellular Ca2 ([Ca2+](o)) to oocytes injected with trpl cRNA and to mock-injected c ontrols, both loaded with the fluorescent Ca2+ indicator fluo-3, induc ed a rapid initial and a slower sustained rate of increase in fluoresc ence, which were designated the initial and sustained rates of Ca2+ in flow respectively. Compared with mock-injected oocytes, trpl-cRNA-inje cted oocytes exhibited a higher resting cytoplasmic free Ca2+-concentr ation ([Ca2+](i)), and higher initial and sustained rates of Ca2+ infl ow in the basal (no agonist) states. The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inf low, (2) an increase in the amount of trpl cRNA injected and (3) an in crease in [Ca2+](o). Gd3+ inhibited the trpl cRNA-induced basal rate o f Ca2+ inflow, with a concentration of approx. 5 mu M Gd3+ giving half -maximal inhibition. Expression of trpl cRNA also caused an increase i n the basal rate of Mn2+ inflow. The increases in resting [Ca2+](i) an d in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and pep tide (281-309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjecti on) activated, and a higher concentration inhibited, the trpl cRNA-ind uced increase in basal rate of Ca2+ inflow. The action of the high con centration of exogenous calmodulin was reversed by W13 and calmodazoli um. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compa red with those in mock-injected oocytes, the guanosine 5'-[beta-thio]d iphosphate-stimulated rate was greater, the onset of thapsigargin-stim ulated initial rate somewhat delayed and the inositol 1,4,5-trisphosph ate-stimulated initial rate markedly inhibited. It is concluded that ( 1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes; (2) in the environment of the Xen opus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is acti vated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be a ctivated by depletion of Ca2+ in the endoplasmic reticulum; and (3) ex pression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activate d Ca2+ channels.