Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca super(2+) channel activated by Ca super(2+) and calmodulin, and by guanosine 5'[ gamma -thio]triphosphate

• Published in: Biochemical journal 1996-01, Vol.316 (3), p.793-803
• Main Authors: Lan, Ling, Bawden, MJ, Auld, A M, Barritt, G J
• Format: Article
• Language: English
• Subjects: Drosophila; Freshwater; Xenopus laevis
• ISSN: 0264-6021

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca super(2+) channel, on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca super(2+) ([Ca super(2+)] sub(o)) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca super(2+) 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 Ca super(2+) inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca super(2+) concentration ([Ca super(2+)] sub(i)), and higher initial and sustained rates of Ca super(2+) inflow in the basal (no agonist) states. The basal rate of Ca super(2+) inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca super(2+) inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca super(2+)] sub(o). Gd super(3+) inhibited the trpl cRNA-induced basal rate of Ca super(2+) inflow, with a concentration of approx. 5 mu M Gd super(3+) giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn super(2+) inflow. The increases in resting [Ca super(2+)] sub(i) and in the basal rate of Ca super(2+) inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281-309) of (Ca super(2+) 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 Ca super(2+) inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca super(2+) 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 Mn super(2+) as well Ca