Injection of rat hepatocyte poly(A) super(+) RNA to Xenopus laevis oocytes leads to expression of a constitutively-active divalent cation channel distinguishable from endogenous receptor-activated channels

• Published in: Cell calcium (Edinburgh) 1996-01, Vol.19 (5), p.439-452
• Main Authors: Auld, A M, Bawden, MJ, Berven, LA, Harland, L, Hughes, B P, Barritt, G J
• Format: Article
• Language: English
• Subjects: Xenopus laevis
• ISSN: 0143-4160

The expression of hepatocyte plasma membrane receptor-activated divalent cation channels in immature (stages V and VI) Xenopus laevis oocytes and the properties which allow these channels to be distinguished from endogenous receptor-activated divalent cation channels were investigated. Divalent cation inflow to oocytes housed in a multiwell plate was measured using the fluorescent dyes Fluo-3 and Fura-2. In control oocytes, ionomycin, cholera toxin, thapsigargin, 3-fluoro-inositol 1,4,5-trisphosphate (InsP sub(3)F) and guanosine 5'-[ gamma -thio]triphosphate (GTP gamma S) stimulated Ca super(2+) and Mn super(2+) inflow following addition of these ions to the oocytes. Ionomycin-, cholera-toxin-, thapsigargin-and InsP sub(3)F-stimulated Ca super(2+) inflow was inhibited by Gd super(3+) (half maximal inhibition at less than 5 mu M Gd super(3+) for InsP sub(3)F-stimulated Ca super(2+) inflow). GTP gamma S-stimulated Ca super(2+) inflow was insensitive to 50 gamma M Gd super(3+) and to SK&F 96365. These results indicate that at least three types of endogenous receptor-activated Ca super(2+) channels can be detected in Xenopus oocytes using Ca super(2+)-sensitive fluorescent dyes: lanthanide-sensitive divalent cation channels activated by intracellular Ca super(2+) store depletion, lanthanide-sensitive divalent cation channels activated by cholera toxin, and lanthanide-insensitive divalent cation channels activated by an unknown trimeric G-protein. Oocytes microinjected with rat hepatocyte poly(A)+ RNA exhibited greater rates of Ca super(2+) and Mn super(2+) inflow in the basal (no agonist) state, greater rates of Ca super(2+) inflow in the presence of vasopressin or InsP sub(3)F and greater rates of Ba super(2+) inflow in the presence of InsP sub(3)F, when compared with 'mock'-injected oocytes. In poly(A) super(+) RNA-injected oocytes, vasopressin- and InsP sub(3)F-stimulated Ca super(2+) inflow, but not basal Ca super(2+) inflow, was inhibited by Gd super(3+). It is concluded that at least one type of hepatocyte plasma membrane divalent cation channel, which admits Mn super(2+) as well as Ca super(2+) and is lanthanide-insensitive, can be expressed and detected in Xenopus oocytes.