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

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 (InsP₃F) and guanosine 5′-[γ-thio]triphosphate (GTPγS) stimulated Ca²⁺ and Mn²⁺ inflow following addition of these ions to the oocytes. Ionomycin-, cholera-toxin-, thapsigargin-and InsP₃F-stimulated Ca²⁺ inflow was inhibited by Gd³⁺ (half maximal inhibition at less than 5 μM Gd³⁺ for InsP₃F-stimulated Ca²⁺ inflow). GTPγS-stimulated Ca²⁺ inflow was insensitive to 50 μM Gd³⁺ and to SK&F 96365. These results indicate that at least three types of endogenous receptor-activated Ca²⁺ channels can be detected in Xenopus oocytes using Ca²⁺-sensitive fluorescent dyes: lanthanide-sensitive divalent cation channels activated by intracellular Ca²⁺ 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 Ca²⁺ and Mn²⁺ inflow in the basal (no agonist) state, greater rates of Ca²⁺ inflow in the presence of vasopressin or InsP₃F and greater rates of Ba²⁺ inflow in the presence of InsP₃F, when compared with 'mock'-injected oocytes. In poly(A)⁺ RNA-injected oocytes, vasopressin- and InsP₃F-stimulated Ca²⁺ inflow, but not basal Ca²⁺ inflow, was inhibited by Gd³⁺. It is concluded that at least one type of hepatocyte plasma membrane divalent cation channel, which admits Mn²⁺ as well as Ca²⁺ and is lanthanide-insensitive, can be expressed and detected in Xenopus oocytes.