Figure 3.

Malfunction of Na⁺/K⁺ pump primary aldosteronism (PA)-inducing mutants. Current recorded at −50 mV in Xenopus oocytes coexpressing the human β subunit with either wild-type α1 (A), delF100-L104-α1 (B), or G99R-α1 (C). The gray dotted line indicates the zero-current level (I = 0). The current in 150 mM external Na⁺ is close to zero for wild type and for G99R, but it is negative (inward) in the oocyte expressing the deletion mutant delF100-L104. In all cases, application of extracellular K⁺ (at the indicated concentrations) induces a positive deflection of the current. In the presence of K⁺, the net current is positive (outward) for wild type and G99R, but it is inward for delF100-L104. Note that the Na⁺/K⁺ pump specific inhibitor ouabain blocks the current deflections induced by subsequent application of K⁺ and eliminated the inward current carried by delF100-L104 mutant. Also, note that the K⁺-induced currents in G99R require higher K⁺ concentrations than wild type. The intracellular Na⁺ concentration of these three oocytes was increased by a 30-min incubation in a sodium-loading solution containing (in mM) 90 NaOH, 20 tetraethylammonium-OH, 0.2 EGTA, and 40 HEPES (pH 7.2 with sulfamic acid). D: oocytes expressing wild type, G99R, or only endogenous pumps (uninjected) were incubated for 15 min in a solution composed of (in mM) 4.5 RbCl (with ⁸⁶RbCl), 133 methane sulfonic acid (MS), 125 mM NaOH, 10 HEPES, 5 Ba(OH)₂, 1 Mg(OH)₂, 0.5 Ca(OH)₂, 0.05 bumetanide (pH 7.4). The experiment was performed without and with 100 µM ouabain to obtain the Na⁺/K⁺ pump-specific uptake of Rb⁺, which is imported like K⁺. Note at normal concentrations of intracellular Na⁺ (i.e., without preincubation in Na⁺-loading solution), the oocytes injected with G99R cRNA were indistinguishable from uninjected oocytes, indicating that the G99R present a loss of function under these physiological conditions. Data in D are the average from independent determinations in the number of oocytes indicated in parenthesis, replotted form Fig. 11B in Ref. 62 with error bars representing SD instead of SE; experiments in A–C are distinct from those previously published, but illustrate the same properties described in Ref. 62. For further experimental details see Ref. 62.