On the effect of hyperaldosteronism-inducing mutations in Na/K pumps

Abstract

Primary aldosteronism, a condition in which too much aldosterone is produced and that leads to hypertension, is often initiated by an aldosterone-producing adenoma within the zona glomerulosa of the adrenal cortex. Somatic mutations of ATP1A1, encoding the Na/K pump α1 subunit, have been found in these adenomas. It has been proposed that a passive inward current transported by several of these mutant pumps is a "gain-of-function" activity that produces membrane depolarization and concomitant increases in aldosterone production. Here, we investigate whether the inward current through mutant Na/K pumps is large enough to induce depolarization of the cells that harbor them. We first investigate inward currents induced by these mutations in Xenopus Na/K pumps expressed in Xenopus oocytes and find that these inward currents are similar in amplitude to wild-type outward Na/K pump currents. Subsequently, we perform a detailed functional evaluation of the human Na/K pump mutants L104R, delF100-L104, V332G, and EETA963S expressed in Xenopus oocytes. By combining two-electrode voltage clamp with [³H]ouabain binding, we measure the turnover rate of these inward currents and compare it to the turnover rate for outward current through wild-type pumps. We find that the turnover rate of the inward current through two of these mutants (EETA963S and L104R) is too small to induce significant cell depolarization. Electrophysiological characterization of another hyperaldosteronism-inducing mutation, G99R, reveals the absence of inward currents under many different conditions, including in the presence of the regulator FXYD1 as well as with mammalian ionic concentrations and body temperatures. Instead, we observe robust outward currents, but with significantly reduced affinities for intracellular Na⁺ and extracellular K⁺ Collectively, our results point to loss-of-function as the common mechanism for the hyperaldosteronism induced by these Na/K pump mutants.

Figure 1.

Location of hyperaldosteronism mutations. Zoomed-in view of the ion-binding sites in the E1(3Na) pig Na/K pump structure (Protein Data Bank accession no. 2ZXE) indicating several ion-coordinating residues. The three Na⁺ ions bound are shown in purple, and the carbon backbone of residues altered by hyperaldosteronism-associated Na/K pump mutations studied here is the same color scheme used for each mutant throughout the article.

Figure 2.

Hyperaldosteronism mutations in Xenopus Na/K pumps. (A) TEVC recording at −50 mV from a Na⁺-loaded oocyte expressing wild type. Application of 10 mM K⁺ in NMG⁺_o stimulated outward current. There is zero ouabain (ouab)–sensitive steady-state current in Na⁺_o alone. (B) A similar TEVC recording from an oocyte expressing L104R, 3 d after cRNA injection. K⁺_o-induced outward current was absent, and switching from NMG⁺_o to Na⁺_o induced an inward current that was partially inhibited by perfusion of 10 mM ouabain. Vertical deflections along the current trace represent 100-ms voltage pulses to obtain I-V curves. (C) Mean ouabain-sensitive I-V plots measured in NMG⁺_o (filled symbols) and in Na⁺_o (open symbols), 3–4 d after injection, from oocytes expressing L104R (down triangles), V332G (circles), delF100-L104 (diamonds), and EETA963S (up triangles). Number of experiments is indicated in parentheses. Error bars represent SEM.

Figure 3.

Effect of D933N on the mutants’ leak currents. (A) Continuous TEVC recording at −50 mV from an oocyte expressing V332G/D933N. (B) Mean I_ouab in NMG⁺_o (filled symbols) and Na⁺_o (open symbols) for double mutants L104R/D933N (down triangles), V332G/D933N (circles), delF100-L104/D933N (diamonds), and EETA963S/D933N (up triangles), recorded 3–4 d after injection. Note axis break at negative currents caused by larger currents in oocytes expressing delF100-L104/D933N. The number of experiments is indicated in parentheses. Error bars represent SEM. (C) Western blot of protein recognized by the Anti-KETYY antibody targeting the C-terminal end of the Na/K-ATPase. Left lane shows purified sheep kidney enzyme (0.5 µg total protein) and a membrane preparation from 25 oocytes injected with Xenopus RD-α1-EETA963S/D933N cRNA (20 µg total protein). Bands at ∼110 kD (the approximate mass of the Na/K-ATPase α-subunit) are visible for both samples.

Figure 4.

Wild-type, EETA963S, and EETA963/D933N human pumps. (A–D) Representative current recordings from Na⁺_o-loaded oocytes that were uninjected (A) or injected with human Na/K pump cRNA encoding wild type (B), EETA963S (C), or EETA963S/D933N (D). Left traces show a continuous recording illustrating the effect of several experimental maneuvers on holding current at −50 mV. In all four cases, initial application of K⁺ in NMG⁺_o activated outward current. Substitution of NMG⁺_o with Na⁺_o induced inward current only in EETA963S. For wild type and EETA963S, 4.5 mM K⁺ applied in Na⁺ activated a large outward current. Note that after a 2-min application of 1 mM ouabain, there is no response to subsequent application of K⁺ in all cases, and that the inward current through EETA963S is irreversibly blocked. Right traces show ouabain-sensitive currents measured in Na⁺_o in the same oocytes shown on the left, evoked by application of 100-ms-long pulses to voltages between −140 and 40 mV in 20-mV increments. Gray dashed lines indicate zero-current level. (E) Mean Q-V curves from uninjected (stars, n = 7), wild-type–injected (squares, n = 5), EETA963S-injected (up triangle, n = 4), and EETA963S/D933N-injected (circles, n = 5) oocytes. (F) Mean ouabain-sensitive, steady-state currents in Na⁺_o for same conditions and oocytes in E. Error bars represent SEM.

Figure 5.

L104R, V332G, and delF100-L104 human pumps. (A–C) Representative current recordings from Na⁺-loaded oocytes, expressing L104R (A), V332G (B), and delF100-L104 (C). Left traces show the effect on holding current at −50 mV of the same experimental maneuvers shown in Fig. 4 C. Right traces show voltage pulse-evoked ouabain-sensitive currents in Na⁺ solution from the same oocytes shown on the left. Gray dashed lines indicate zero-current level.

Figure 6.

Ouabain-sensitive currents in wild-type and mutant human Na/K pumps. (A–E) Mean ouabain-sensitive I-V plots in different ionic conditions from oocytes expressing wild-type (n = 5; A), L104R (n = 5; B), V332G (n = 5; C), delF100-L104 (n = 5; D), and EETA963S (n = 4; E) pumps, recorded 2–4 d after injection in experiments similar to those in Figs. 4 and 5. Shown are ouabain-sensitive currents in NMG⁺_o (circles), NMG⁺_o + 3 mM K⁺_o (up triangles), Na⁺_o (squares), and Na⁺_o + 4.5 mM K⁺_o (down triangles; symbol key in A). The insets in B–D show a zoomed-in view of the axes to illustrate the shift in reversal potential (V_REV) when Na⁺_o was replaced with NMG⁺_o. (F) Mean reversal potential of ouabain-sensitive currents in NMG⁺_o (solid bars) and Na⁺_o (striped bars). Error bars represent SEM.

Figure 7.

Turnover rates of wild-type and mutant human Na/K pumps. (A) Representative recording from an oocyte expressing wild-type pumps, held at −50 mV, in which application of 3 mM K⁺_o in NMG⁺_o induced outward current. (B) Representative recording from an oocyte expressing L104R pumps in which replacing NMG⁺_o with Na⁺_o solution induced inward current. The quantity of [³H]ouabain bound to the oocytes in A and B is also indicated. (C) Bar graph showing mean turnover rates (moles of charge per second/moles of [³H]ouabain bound; i.e., s⁻¹) measured in individual oocytes, for the outward current (bracketed as “OUT”) in oocytes expressing wild type (n = 17) and for the Na⁺_o-induced inward current (bracketed as “IN”) in oocytes expressing L104R, V332G, delF100-L104, and EETA963S. Measurements were performed 4–5 d after injection, except for delF100-L104, which was performed 3 d after injection because of large currents. Note the break along the y axis. The number of experiments is indicated in parentheses. Error bars represent SEM.

Figure 8.

K⁺_o dependence of wild-type and G99R pumps. (A and B) TEVC recordings at −50 mV from representative oocytes 4–5 d after injection with wild-type (A) or G99R (B) cRNA. Application of K⁺_o in Na⁺_o solution stimulated outward current in a [K⁺_o]-dependent manner. Addition of 10 and 20 mM K⁺_o did not activate outward current after application and withdrawal of 1 mM ouabain. Insets in A and B are the expanded time-scale ouabain-sensitive transient currents in Na⁺_o upon 100-ms voltage steps from −50 mV. (C) Mean K_0.5 for K⁺_o, as a function of voltage, for wild type (squares) and G99R (circles), measured in 125 mM Na⁺_o (open symbols) or 150 mM Na⁺_o (solid symbols). Wild type at 125 mM Na⁺_o (n = 6) and at 150 mM Na⁺_o (n = 4); G99R at 125 mM Na⁺_o (n = 3) and at 150 mM Na⁺_o (n = 8). (D) Mean normalized Q-V curves in 125 mM Na⁺_o for wild type (squares, n = 7) and G99R (circles, n = 6). Line plots represent a Boltzmann with parameters in the text. Error bars in C and D are SEM, smaller than the symbols for most data points.

Figure 9.

Function of G99R in 150 mM Na⁺_o, with FXYD1 and at 34°C. (A) Continuous recording at −50 mV from an oocyte injected with G99R in which increasing K⁺_o concentrations are applied in the presence of 150 mM Na⁺_o (K_0.5-V plotted in Fig. 8). The ouabain-sensitive currents in Na⁺_o elicited by voltage pulses from −180 to 40 mV, in 20-mV increments, are shown in high temporal resolution in the center. The Q-V curve from those transient currents are shown on the right and was fitted with a Boltzmann distribution (line plot) with parameters Q_tot = 33.4 nC, V_1/2 = −108 mV, and k = 53 mV. All eight oocytes gave comparable results with inward currents absent from recordings. (B) Current at −50 mV from an oocyte injected with α1-G99Rβ1FXYD1 to which increasing K⁺_o concentrations were applied in the 125 mM Na⁺_o. The transient currents elicited are shown in the center and the Q-V curve from those transient currents are on the right. The Boltzmann distribution (fitted line plot) had parameters Q_tot = 12.1 nC, V_1/2= −136 mV, and k= 77 mV. Note that despite robust expression demonstrated by a large Q_tot, the total pump current is largely reduced compared with G99R without FXYD1, even at 20 mM K⁺_o. Three oocytes gave similar results with lower pump currents than non-FXYD1 coinjected oocytes. (C) Recording at −50 mV from an oocyte injected with G99R in which application of 5 mM K⁺_o in 125 mM Na⁺_o was performed at 24°C and then at 34°C. Note absence of significant inward current upon application of ouabain, despite deterioration of the oocyte. Three oocytes gave nearly identical results.

Figure 10.

Na⁺_i dependence of wild-type and G99R pumps. (A and B) Representative current recordings at 0 mV from giant inside-out patches excised from an oocyte expressing wild type (A) or one expressing G99R (B). The patches were perfused with intracellular solutions of varying [Na⁺_i] (a mix of Na⁺_i and K⁺_i solutions; Materials and methods). Application of 4 mM ATP_i induced outward pump currents in a [Na⁺_i]-dependent manner. Vertical deflections represent 25-ms voltage steps. (C) Mean [Na⁺_i]-dependence of ATP-induced currents normalized to the I_max from Hill fits to the mean data for wild type (squares, n = 5) and G99R (circles, n = 4) with parameters K_0.5 = 13.1 mM, n_H = 2.9 for wild type and K_0.5 = 33.1 mM, n_H = 1.2 for G99R (mean from fits in individual experiments are shown in the text). Mean ATP-induced current was 12.4 ± 2.6 pA in 50 mM Na⁺_i 90 mM K⁺_i for wild type and 12.4 ± 1.3 pA in 125 mM Na⁺_I for G99R.

Figure 11.

Radioactive ⁸⁶Rb⁺ and ²²Na⁺ uptake in oocytes expressing human Na/K pumps. (A) Mean ⁸⁶Rb⁺ uptake in Na⁺-loaded oocytes, 4 d after injection, during 5-min incubation in 125 mM Na⁺_o with 4.5 mM Rb⁺_o, either in the absence (open bars) or presence (striped bars) of 100 µM ouabain. (B) Mean ⁸⁶Rb⁺ uptake in oocytes, 4 d after injection, which were not Na⁺ loaded, during 15-min incubations in the same ionic conditions as in A. In both A and B, ⁸⁶Rb⁺ uptake was also measured in uninjected oocytes from the same batches. (C) Mean ²²Na⁺ uptake during a 2-h incubation in 125 mM Na⁺_o by oocytes expressing wild-type or mutant pumps, 4–5 d after injection in the absence (open bars) or presence (striped bars) of 100 µM ouabain. The number of oocytes is indicated in parentheses above each column. Error bars represent SEM.