Spontarelli K , Infield DT , Nielsen HN , Holm R , Young VC , Galpin JD , Ahern CA , Vilsen B , Artigas P .

MCB-1515434 National Science Foundation, R24 NS104617 NINDS NIH HHS , R223-2016-595 Lundbeck Foundation, 7016-00193B Danish Council For Independent Research, R03 NS116433 NINDS NIH HHS

             potassium ion transmembrane transport

	Figure 1. Relevant functional and structural properties of the Na + /K + pump. (A) Post-Albers kinetic scheme describing partial reactions in the Na+/K+ pump cycle. (B) Overall view of the Na+/K+ pump structure in E1(3Na+) form, PDB accession no. 3WGV. The boxed part shows the ion-binding region (with three Na+ ions as blue spheres; see further below). In the cytoplasmic region, the nucleotide, phosphate analog (both in blue stick representation) and phosphorylated aspartate (stick representation in color by elements) are shown. (C) Zoomed-in view of the ion-binding region of the E1(3Na+) structure showing amino-acid side chains participating in ion coordination, as well as Y780 and surrounding residues (stick representation in color by elements). Broken lines indicate potential hydrogen bonds from Y780 (2.77 indicates the length in A of the bond between Y780 hydroxyl and T816 backbone oxygen). Numbers on the Na+-ions (blue spheres) are the ion-binding site number. (D) Zoomed-in view of the ion binding region of E2Pi(2K+) structure (PDB accession no. 2ZXE) with the same residues shown. K+ ions and a water molecule are shown as purple spheres and a red sphere, respectively. Broken lines indicate potential hydrogen bonds from Y780 (2.73 indicates the length in A of the bond between Y780 hydroxyl and D817 side-chain oxygen). Note that Y780 exchanges its hydrogen-bonding partner from T816 to D817 in connection with the E1–E2 conformational change (in E2 the length between Y780 hydroxyl and T816 backbone oxygen has increased to 4.95 A). (E) Amino-acid sequence alignment of the catalytic subunit of P-type-ATPases showing high conservation of the tyrosine residue studied in this article. P2-ATPaseses shown are Na+,K+-ATPase α1 subunit (NKA), the gastric H+,K+-ATPase α subunit (HKA), the plasma membrane Ca2+-ATPase catalytic subunit (PMCA), and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase catalytic subunit (SERCA). Other P-type ATPases indicated are the Cu-ATPase ATP7B (P1-ATPase), the H+-ATPase from Arabidopsis thaliana (P3-ATPase), and the human flippase ATP8A1 (P4-ATPase).
	Figure 2. Representative LRMS (± mode) of Phe-pdCpA. Calculated mass for C28H35N9O14P2, 783.18 D.
	Figure 3. Interaction with extracellular ions of WT and Y780F. (A) Representative currents at −50 mV from oocytes expressing WT and Y780F mutant. Application of K+ in NMDG+ external solution at the millimolar concentrations indicated over the grey lines activates Na+/K+ pump current. At −50 mV all current induced by ≤10 mM K+ is Na+/K+ pump current (Meyer et al., 2020; Stanley et al., 2015). (B) Pump current amplitude induced by 10 mM K+ in NMDG+, number of oocytes indicated in parentheses. (C) K0.5.K from Hill plots of dose-response data in NMDG+ and in 125 mM Na+. Individual data points are shown as grey spheres. (D and E) Representative ouabain-sensitive currents from two oocytes expressing WT (D) or Y780F (E). The currents were elicited by the pulse protocol illustrated in the box in two conditions: 125 mM Na+ (top) and 62.5 mM Na+ (bottom). The pulse protocol was repeated in the absence and then in the presence of ouabain, to obtain the subtracted ouabain-sensitive current displayed. (F and G) Q–V curves for the integral of the currents during the pulse (QON), at 125 mM Na+, and upon return to −50 mV (QOFF) at 125 mM Na+ (solid) and 62.5 mM Na+ (open) for WT (F) and Y780F (G) expressing oocytes displayed in D and E. Line plots are fits by Boltzmann distribution (Eq. 4) with parameters V1/2 = −49.1 mV, kT/zqe = 43.6 mV, Qtot = 39.7 nC (QON at 125 mM Na+), V1/2 = −38.2 mV, kT/zqe = 39.1 mV, Qtot = 35.7 nC (QOFF at 125 mM Na+) and V1/2 = −60.5 mV, kT/zqe = 39.1 mV, Qtot = 34 0.1 nC (QOFF at 62.5 mM Na+) for WT, and V1/2 = −119.6 mV, kT/zqe = 56.8 mV, Qtot = 16.8 nC (ON at 125 mM Na+, fit to −160 mV only), V1/2 = −121 mV kT/zqe = 48 mV Qtot = 16.3 nC (OFF at 125 mM Na+) and V1/2 = −131 mV kT/zqe = 48 mV Qtot = 14.5 nC (OFF at 62.5 mM Na+), for Y780F. (H) Mean QOFF−V curves (normalized to Qtot from Boltzmann equations) from oocytes expressing WT (black, n = 5) or Y780F (red, n = 8) where transient charge movement was evaluated in both 125 and 62.5 mM Na+. Line plots are Boltzmann curves with parameters: V1/2 = −42.0 mV, kT/zqe = 35 mV at 125 mM Na+ and V1/2 = −67.6 mV, kT/zqe = 35 mV for WT and V1/2 = −113.5 mV, kT/zqe = 51.8 mV at 125 mM Na+ and V1/2 = −144.8 mV, kT/zqe = 51.8 mV at 62.5 mM Na+, for Y780F. A shared slope factor (kT/zqe) was used for the two concentrations in each mutant.
	Figure 4. Interaction with intracellular Na + and ATPase activity of WT and Y780F. (A) ATP induced currents at 0 mV from two patches excised from two oocytes: one expressing WT pumps (top, black traces) and another expressing Y780F pumps (bottom, red traces). The patch pipettes contained extracellular NMDG+ solution with 5 mM K+ and the bath intracellular solution (NMDG+ + Na+ = 125 mM) with the indicated Na+ concentration. (B) Mean normalized ATP-activated Na+/K+ pump currents from three patches as a function of the intracellular Na+ concentration. Lines are fits of a Hill equation, with best fit parameters K0.5 = 3.47 ± 0.25 mM, nH = 1.4 ± 0.1 for WT and K0.5 = 17.9 ± 0.96 mM, nH = 1.9 ± 0.2 for Y780F. (C) Na+ dependence of phosphorylation. Phosphorylation was carried out for 10 s at 0°C with 2 μM [γ-32P]ATP in 20 mM Tris (pH 7.5), 3 mM MgCl2, 1 mM EGTA, 10 µM ouabain, 20 µg oligomycin/ml, and the indicated concentration of Na+ added as NaCl with various concentration of NMDG+ to maintain constant ionic strength. Line plots represent the best fit of a Hill function (Eq. 1 in Materials and methods) with K0.5 ± SD and the number of independent experiments reported in Table 1. (D) K+ dependence of Na+,K+-ATPase activity determined at 37°C in 40 mM NaCl, 3 mM ATP, 3 mM MgCl2, 30 mM histidine (pH 7.4), 1 mM EGTA, 10 µM ouabain, and the indicated concentration of K+ added as KCl. Line plots represent the best fit of a double Hill function to the data (see Materials and methods), with K0.5 ± SD and the number of independent experiments corresponding to the rising part reported in Table 1. For C and D, error bars (seen only when larger than the size of the symbols) represent SEM. (E) Turnover rate of WT and Y780F (mean ± SD) calculated as the ratio between the maximum ATPase activity (determined at 130 mM Na+ and 20 mM K+ under conditions otherwise similar to those for D) and the active site concentration (phosphorylation level obtained under stoichiometric conditions, i.e., as for C at 150 mM NaCl; Nielsen et al., 2019).
	Figure 5. Interaction of Y780L/Q/A with extracellular ions. (A) Na+/K+ pump current induced by 10 mM K+ in NMDG+. (B) K0.5.K obtained from Hill fits (Eq. 2) to the K+-induced current at −50 mV in NMDG+ or with 125 mM Na+. Grey circles show each independent measurement in individual oocytes. (C) Representative ouabain-sensitive currents elicited by 50-ms-long pulses from −50 mV to the indicated voltages, in oocytes bathed by 125 mM Na+ solution, expressing Y780L, Y780Q, or Y780A. (D) Mean QOFF–V curve from 6 to 12 experiments. Continuous lines are Boltzmann distribution fits to the average data, with kT/zqe = 36 mV and Qtot = 6.65 nC for WT and kT/zqe = 50 mV; Qtot = 7.32 nC for Y780F, kT/zqe = 45 mV, Qtot = 4.24 nC for Y780Q and kT/zqe = 60 mV, Qtot = 2.24 nC (Y780A). The mean V1/2 from individual experiments is listed in Table 1. The inset shows normalized Boltzmann equations to illustrate shifts in Q–V, except for Y780L which could not be fit, see Results.
	Figure 6. Ion concentration dependence of phosphorylation and ATPase activity. (A) Na+ dependence of phosphorylation determined as described in Fig. 4 C. Line plots represent the best fit of a Hill function (Eq. 1) with K0.5 ± SD and number of independent experiments reported in Table 1. (B) K+ dependence of Na+,K+-ATPase activity determined as in Fig. 4 D. Line plots represent the best fit of a double Hill function to the data (see Materials and methods), with K0.5 ± SD and number of independent experiments corresponding to the rising part reported in Table 1. (C) K+ affinity determined by K+ inhibition of phosphorylation. Phosphorylation was carried out as in A, with 100 mM NaCl and the indicated concentration of K+ added as KCl (with various concentrations of choline chloride to maintain a constant ionic strength), but without oligomycin. Line plots represent the best fit of a negative Hill function (Eq. 2). The extracted K0.5 values ± SD and the number of independent experiments are reported in Table 1. For all panels, error bars (seen only when larger than the size of the symbols) represent SEM.
	Figure 7. E1P(3Na + )–E2P distribution evaluated by ADP sensitivity and E2(2K + ) → E1 conformational transition evaluated by ATP dependence of Na + ,K + -ATPase activity. (A) ADP-dependent dephosphorylation. Phosphorylation was carried out for 5 s at 0°C with 2 µM [γ-32P]ATP in 20 mM Tris (pH 7.5), 100 mM NaCl, 50 mM choline chloride, 3 mM MgCl2, 1 mM EGTA, and 10 µM ouabain. Dephosphorylation was initiated by the addition of 2.5 mM ADP and 1 mM unlabeled ATP. The dephosphorylation reaction was terminated by acid quenching after the indicated time intervals. Line plots represent the best fit of a bi-exponential decay function (Eq. 3 in Materials and methods) with the rate constant corresponding to the rapid phase, reflecting the ADP reaction with E1P, set to 2 s−1. The relative fraction of E2P together with the rate constant corresponding to the slow phase, reflecting E2P dephosphorylation, are indicated in Table 1 as mean ± SD and number of independent experiments. (B) ATP dependence of Na+,K+-ATPase activity. ATPase activity was determined at 37°C in 130 mM NaCl, 20 mM KCl, 3 mM MgCl2, 30 mM histidine (pH 7.4), 1 mM EGTA, 10 µM ouabain, and the indicated ATP concentrations. Line plots represent the best fit of a Hill function (Eq. 1 in Materials and methods). K0.5 values (in µM ± SD) and number of experiments (n) in parentheses are: WT, 402 ± 74 (12); Y780A, 63 ± 20 (6); Y780L, 362 ± 122 (4); Y780F, 368 ± 19 (4); Y780Q, 121 ± 61 (7). In all panels, symbols are mean ± SEM (seen only when larger than the symbols). For each assay, the WT data from the left panel is reproduced as broken lines in the other panels.
	Figure 8. Electrophysiological characteristics of Y780C, Y780T, Y780S, and Y780H. (A) Ouabain-sensitive currents in 125 mM Na+, elicited by 50 ms pulses to the indicated voltages. (B) Mean Q–V curve, obtained from the integral of the transient currents when the voltage returns to −50 mV, for WT, Y780C, Y780T, Y780S, and Y780H. (C) Normalized fitted curves from B, to illustrate changes in the Boltzmann distributions fitted to the mean data, with centers V1/2 = −54 mV for WT, V1/2 = −75 mV for Y780C, V1/2 = −98 mV for Y780S, V1/2 = −101 mV for Y780T, and V1/2 = −104 mV for Y780H. WT and Y780F are shown as black and red dotted lines, respectively. (D and E) K0.5 for K+ activation of pump current when applied in NMDG+ external solution (D) or Na+ solution (E). Grey circles show individual data points from each oocyte. (F) Current–voltage relationship for the ouabain-sensitive currents in 125 mM Na+ at four K+ concentrations, for WT (left, n = 12) and the disease-causing Y780C (center, n = 2) and Y780H (right, n = 7). The dotted line marks the 0-current level.
	Figure 9. Feasibility of non-sense suppression to express Na + /K + pumps. (A) Mean current induced by 10 mM K+ 3 or 4 d after injection with either WT cRNA or Y780tag mutant cRNA + Tyr-tRNA (NSS-Tyr). The same batches of oocytes were tested on days 3 and 4 after injection. The pump current in oocytes expressing NSS-Tyr was smaller than WT, whether 3 or 4 d after injection. (B) Mean current induced by 10 mM K+ in the absence of Na+ (left) and apparent dissociation constant K0.5.K (right) from Hill fits in dose–response curves for K+ activation at −50 mV in NMDG+ 4 d after injection. Grey circles show individual data points in different oocytes. (C) Mean QOFF–V curves measured (125 mM Na+, 0 K+) for uninjected oocytes and for oocytes injected with WT cRNA, Y780F cRNA, Y780tag cRNA + Tyr-tRNA (NSS-Tyr), or Y780tag cRNA + Phe-tRNA (NSS-Phe). The line plots are the fits to Boltzmann distributions. The inset shows the mean normalized curves illustrating identical voltage dependencies for both pairs: WT NSS-Tyr and Y780F NSS-Phe.
	Figure 10. K + -activated pump current with unnatural amino acids at position 780. (A) Mean 10 mM K+-induced current in oocytes injected with Y780tag cRNA and tRNAs for tyrosine (NSS-Tyr), phenylalanine (NSS-Phe), and other phenylalanine derivatives. The label pdCpA designates the full-length ligated (but unacylated) tRNA control (see Materials and methods). (B) Mean K0.5.K for substitutions with large enough pump currents. Grey circles show individual data points in different oocytes. The chemical structure of the amino acids is shown on top of the bars and the error bars represent the SEM.
	Figure 11. Transient charge movement with unnatural amino acids at 780. (A) Ouabain-sensitive traces elicited by applying pulses from −50 mV to the indicated voltages in oocytes either uninjected or injected using the non-sense suppression method with the indicated natural and unnatural amino acids. The current scale is identical for all traces. Time scale is different only for di-m-F-Phe (2F-Phe) for which pulses were 50 ms instead of 100 ms long. (B) Mean QOFF–V curve from experiments like those in A, number of experiments (four to nine) given with mean parameters from individual experiments in the text. The line plots are fits to Boltzmann distributions (Eq. 4) to the average data with parameters: V1/2 = −45.6 mV, kT/zqe = 35.0 mV, Qtot = 1.91 nC for NSS-Tyr (Tyr); V1/2 = −136 mV, kT/zqe = 55.9 mV, Qtot = 2.65 nC for NSS-Phe (Phe); V1/2 = −46.7 mV, kT/zqe = 40.3 mV, V1/2 = −166 mV, kT/zqe = 40 mV, Qtot = 0.97 nC for p-F-Phe (F-Phe). Qtot = 1.27 nC for m-F-Tyr (F-Tyr) V1/2 = −141 mV, kT/zqe = 52.0 mV, Qtot = 0.68 nC for di-m-F-Phe (2F-Phe). Error bars represent the SEM. (C) Boltzmann fits to the average data, normalized to illustrate distinct voltage dependencies with parameters given in the text.

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