The effect of various mutations on the activation of sodium ion/potassium ion pump current by external potassium ion in Xenopus oocytes

A method was developed to measure Na/K pump current from a single functional alpha/beta dimeric form of the pump expressed in Xenopus laevis oocytes. Current from a known combination of alpha and beta subunits can be measured by blocking endogenous pump current with ouabain. The known combination of alpha and beta subunits is expressed by injecting cRNA coding for a ouabain insensitive a subunit and the endogenous oocyte beta subunit. The Ki for dihydroouabain (DHO) inhibition for the a subunit mutations N120R and A121K was 15 +/- 1.8 muM. This is 150 fold greater than that of the endogenous oocyte pump (<0.1 muM). 5' and 3' RACE reactions were used to clone the adult Xenopus laevis brain and oocyte beta3 subunit. Compared to the previously published sequence of the beta3 subunit from embryonic brain, three sequence differences were found in the ectodomain of the oocyte and the adult brain protein, T99, N206S and S241L. The 3' RACE product produced three bands on an agarose gel which when sequenced individually was shown to have four AATAAA polyadenylation sites. The magnitude of K+-induced and ouabain sensitive currents in batches of non injected oocytes was 10 +/- 10 nA. In cells injected with cRNA coding for either the betaoocyte (4 ng/32mul) peptide alone, the magnitude of the pump current was 40 +/- 6 nA. In oocytes co-injected with cRNA coding for either alpha kidney/betaoocyte or the RK2/betaoocyte subunits (20ng/4ng ratio/32mul) the magnitude of the ouabain sensitive current was 181 +/- 25 nA and 179 +/- 22 nA respectively. This demonstrated that the beta subunit gene cloned from oocytes codes for a functional protein that can assemble with protein coded for by exogenously injected cRNA from both the RK2 mutant and the kidney alpha subunit. The lower sensitivity to DHO of the RK2 mutant compared to the native oocyte Na-pump allows us to work in the presence of DHO thus inhibiting the endogenous Na-pump and ensuring that the signal measured is produced by pump coded for by the injected exogenous cRNA. (Abstract shortened by UMI.).