Functional expression of the amiloride-sensitive sodium channel in Xenopus oocytes

Expression of the amiloride-sensitive sodium channel was examined in Xenopus oocytes that were microinjected with A6 cell mRNA. Amiloride-inhibitable ²²Na flux could be measured in intact oocytes 2-3 days after injection with 25 ng of poly(A)⁺ RNA isolated from aldosterone-treated A6 cells. The rate of ²²Na uptake was ~15-fold greater in oocytes microinjected with 25 ng of poly(A)⁺ RNA than in water-injected control oocytes. An increase in ²²Na uptake by mRNA-injected oocytes occurred whether the mRNA was isolated from A6 cells grown on a porous or nonporous support. In the presence of 4 mM NaCl, amiloride caused dose-dependent inhibition of ²²Na uptake in mRNA-injected oocytes, which was half-maximal at 6 x 10^-8 M. Both 1 μM amiloride and 0.1 μM benzamil inhibited ²²Na uptake in mRNA-injected oocytes by > 95%, whereas <50% inhibition occurred with 1 μM 5-(N-ethyl-N-isopropyl)amiloride. When A6 cell mRNA was size fractionated by sucrose density-gradient centrifugation, amiloride-sensitive ²²Na uptake was expressed predominantly by oocytes injected with mRNA from two contiguous fractions.