|
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 â50 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)âsensitive 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), 3â4 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 mutantsâ leak currents. (A) Continuous TEVC recording at â50 mV from an oocyte expressing V332G/D933N. (B) Mean Iouab 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 3â4 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 â¼110 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. (AâD) 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 â50 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 â140 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-typeâinjected (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. (AâC) 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 â50 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. (AâE) 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 2â4 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 BâD show a zoomed-in view of the axes to illustrate the shift in reversal potential (VREV) 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 â50 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., sâ1) measured in individual oocytes, for the outward current (bracketed as âOUTâ) in oocytes expressing wild type (n = 17) and for the Na+o-induced inward current (bracketed as âINâ) in oocytes expressing L104R, V332G, delF100-L104, and EETA963S. Measurements were performed 4â5 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 â50 mV from representative oocytes 4â5 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 â50 mV. (C) Mean K0.5 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 â50 mV from an oocyte injected with G99R in which increasing K+o concentrations are applied in the presence of 150 mM Na+o (K0.5-V plotted in Fig. 8). The ouabain-sensitive currents in Na+o elicited by voltage pulses from â180 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 Qtot = 33.4 nC, V1/2 = â108 mV, and k = 53 mV. All eight oocytes gave comparable results with inward currents absent from recordings. (B) Current at â50 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 Qtot = 12.1 nC, V1/2= â136 mV, and k= 77 mV. Note that despite robust expression demonstrated by a large Qtot, 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 â50 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 ATPi 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 Imax from Hill fits to the mean data for wild type (squares, n = 5) and G99R (circles, n = 4) with parameters K0.5 = 13.1 mM, nH = 2.9 for wild type and K0.5 = 33.1 mM, nH = 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, 4â5 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.
|
