Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5'[gamma-thio]triphosphate.
The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull and Kelly (1992) Neuron 8, 631-642], on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ ([Ca2+]0) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ([Ca2+]i), and higher initial and sustained rates of Ca2+ inflow 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+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca2+]0. Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx. 5 microM Gd3+ giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow. The increases in resting [Ca2+]1 and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281-309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5'-[beta-thio]diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-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 Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels.
Lan
L
L
Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia.
Bawden
M J
MJ
Auld
A M
AM
Barritt
G J
GJ
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D015220:Calcium Channels / Q000096:biosynthesis* / Q000187:drug effects / Q000502:physiology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002147:Calmodulin / Q000494:pharmacology*; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000502:physiology; D003001:Cloning, Molecular; D004330:Drosophila / Q000378:metabolism*; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D005260:Female; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007295:Inositol Phosphates / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D018075:RNA, Complementary / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D050051:Transient Receptor Potential Channels; D014982:Xenopus laevis
D015220:Calcium Channels; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D008565:Membrane Proteins; D018075:RNA, Complementary; D011994:Recombinant Proteins; D013729:Terpenes; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008345:Manganese; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium