Figure 2.

Functional Characterization of Mutant ATP1A1 in Transfected Cells

Mutations corresponding to human ATP1A1 p.Leu302Arg (p.L302R), p.Gly303Arg (p.G303R), and p.Met859Arg (p.M859R) were introduced in rat α1 Na⁺, K⁺-ATPase (rat Atp1a1), which is insensitive to the inhibitor ouabain.

(A) Post-Albers scheme of the Na⁺, K⁺-ATPase reaction cycle.

(B) Transient expression of mutant enzymes with concomitant siRNA-mediated knockdown of endogenous Na⁺, K⁺-ATPase. All three mutants were phosphorylated with [γ-³²P]-ATP in the Na⁺ reaction, indicating expression of mutant proteins and the ability to perform the Na⁺ limb of the Post-Albers reaction cycle (maximal phosphorylation signals relative to WT), albeit at a significantly reduced expression and/or phosphorylation level relative to that of WT, for p.Gly303Arg and p.Met859Arg (“o” = p < 0.001 for p.Gly303Arg and p.Met859Arg by a one-way ANOVA test; p = 0.027 for p.Leu302Arg; n = 3–5).

(C) Na⁺ dependence of phosphorylation showing a significant 3.5-fold reduced affinity for Na⁺ for p.Leu302Arg relative to WT, whereas the affinity was WT-like for p.Gly303Arg and p.Met859Arg, but with reduced cooperativity (see curves separated in different panels with statistics in Figure S1).

(D) K⁺-sensitivity of the Na⁺, K⁺-ATPase phosphoenzyme intermediate. Symbols are the same as in (C). K⁺ interaction was assessed by the ability of K⁺ to inhibit phosphorylation. The Hill equation for inhibition was used for data fitting.⁶ The cooperativity was WT-like for p.Leu302Arg (Hill coefficient 1.3–1.4), whereas the apparent affinity for K⁺ of this mutant was reduced significantly (p < 0.001 by a one-way ANOVA test, n = 8), 36-fold relative to WT. For p.Gly303Arg and p.Met859Arg, the Hill coefficients were only 0.6–0.7, indicating loss of cooperativity between the two K⁺ sites.

(E) Whole-cell currents of adrenal NCI-H295R cells expressing wild-type (WT) or different mutant (p.Leu302Arg, p.Gly303Arg, p.Met859Arg) ouabain-insensitive versions of rat Atp1a1. Compared to WT cells, mutant Atp1a1-expressing cells (except for mutant p.Met859Arg) displayed an abnormal current which was reduced after removal of Na⁺, indicating an abnormal Na⁺ permeability as causative for abnormal inward currents of Na⁺ ions in mutant cells.

(F) Mutant-expressing NCI-H295R cells (except for mutant p.Met859Arg) had a depolarized membrane potential under control conditions but were hyperpolarized to the level of WT cells after removal of extracellular Na⁺.

(G) Intracellular Na⁺ and K⁺ contents in cell lysates of HEK293 cells under control conditions and after treatment with the Na⁺, K⁺-ATPase inhibitor ouabain. Ouabain treatment strongly increased intracellular Na⁺ and decreased intracellular K⁺ in non-transfected cells. Expression of WT rat Atp1a1 significantly attenuated these changes of intracellular Na⁺ and K⁺, whereas this was not the case for all mutant-expressing cells. Expression of the p.Gly303Arg mutant increased Na⁺ and decreased K⁺ even more in comparison to vector control cells. n = 7–9 per group.

(B–G) (all data are presented as means ± SEM).