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 approximately 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 microM amiloride and 0.1 microM benzamil inhibited 22Na uptake in mRNA-injected oocytes by greater than 95%, whereas less than 50% inhibition occurred with 1 microM 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.
George
A L
AL
Department of Medicine, University of Pennsylvania, Philadelphia 19104.
Staub
O
O
Geering
K
K
Rossier
B C
BC
Kleyman
T R
TR
Kraehenbuhl
J P
JP
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D005260:Female; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics*; D008845:Microinjections; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D014982:Xenopus laevis
D008565:Membrane Proteins; D012333:RNA, Messenger; D015222:Sodium Channels; D000584:Amiloride; D012964:Sodium