Vedovato N , Gadsby DC .

R01 HL036783 NHLBI NIH HHS , HL36783 NHLBI NIH HHS

	Figure 1. The Post–Albers transport cycle (e.g., Post et al., 1972) of the Na+,K+-ATPase cartooned as an ion channel with two gates. The gates open strictly alternately to allow access to cation-binding sites from only one side at a time. The extracellular-side gate (red) is closed in all E1 states, and the cytoplasmic-side gate (blue) is closed in E2 states. Parentheses denote occluded ions, and “-P” symbolizes the aspartyl-phosphate formed by autophosphorylation of E1 pumps once three cytoplasmic Na+ ions have bound.
	Figure 2. Outward and inward current components in wild-type Na+/K+ pumps. (A) Current in a single Na+-loaded oocyte overexpressing wild-type α1/β3 Xenopus Na+/K+-ATPase pumps, at −20 mV, in 125-mM Na+o solutions. The addition of 15 mM K+o (bar labeled “K”) activated large outward 3 Na+i/2 K+o exchange current that was abolished by 10 mM ouabain (bar labeled “ouab”). Vertical lines are responses to 50-ms jumps to other potentials. (B) Steady currents from the oocyte in A plotted against voltage for the trials identified by numbers 1–4 in A. (C) Average ouabain-sensitive currents (I ouab-sens norm) obtained by subtraction of currents like those in B, at every voltage and presence or absence of K+o, were normalized to the mean amplitude between 0 and 60 mV of ouabain-sensitive current at 15 mM [K+o] in 0 or 125 mM Na+o, as appropriate; normalized currents were then averaged across oocytes; ±SEM is visible where larger than the symbols; n = 8 at 125 mM Na+o; n = 5 at 0 Na+o. In direct comparisons in the same oocyte, maximal outward ouabain-sensitive current at 15 mM [K+o] and positive voltage is the same in the presence or absence of Na+o (Vedovato and Gadsby, 2010). (D) Ouabain-sensitive currents in a representative oocyte at 0 mM Na+o. At pHo 7.6, ouabain-sensitive currents in 15 mM K+o (closed triangles) or in 0 mM K+o (closed circles) are like those in C, but upon lowering pHo to 6.0 in 0 mM K+o (open circles), inward current at negative potentials increased approximately fivefold.
	Figure 3. Proposed proton transport mechanism and candidate carboxylates. (A) Cartoon of proposed mechanism indicating protonation/deprotonation of a side chain (red circle) with alternate extracellular and cytoplasmic access in kinetically adjacent pump conformations; gray mass represents protein barrier to proton diffusion in the relevant segment of Na+/K+ pump TM domain structure. (B; from left to right) Increasingly magnified (factors indicated by blue arrows) views of Xenopus α1/β3 Na+/K+ pump homology model based on x-ray crystal structure of the K+-bound E2 · MgF42− Na+/K+-ATPase (Protein Data Bank accession no. 2ZXE; Shinoda et al., 2009), showing the α subunit (gray, with 5 of the 10 TM helices colored), β subunit (green surface), and γ subunit (red helix). (Middle and right) Side and top (from extracellular surface) views of the α-subunit TM domain (helix numbers in white) identifying six candidate carboxylates, three Glu (red spheres) and three Asp (green spheres), at the level of binding sites I and II that contain K+ ions (black balls); the colored TM helices are: pale blue, TM1; magenta, TM2; blue, TM4; purple, TM5; and green, TM6. Gray, TM3 and TM7–TM10; lime spheres, C113 in TM1; yellow spheres (left) or sticks (right) mark T806 at the top of TM6.
	Figure 4. External pH sensitivity of ouabain-sensitive inward current in 0 Na+o and 0 K+o after conservative mutation of each candidate residue in partially ouabain-resistant C113Y pumps. Each Glu (red circles) and Asp (green circles) was neutralized, and the inward current response to lowering pHo (open vs. closed circles) was compared with that of the parent C113Y pumps (black circles). For these comparisons, inward current was normalized to maximally K+o-activated (10–30 mM [K+o]), ouabain-sensitive outward pump current (Figs. S1 and S2) recorded in the same oocyte, averaged between 0 and +60 mV; because E336Q and D813N pumps lacked K+o-activated current (Fig. S1, B and D), inward current was normalized to total pump-mediated Na+ charge movement, Qtot, in 0 K+, 125 mM Na+o solution (Nakao and Gadsby, 1986; Vasilyev et al., 2004; Meier et al., 2010; Poulsen et al., 2010; Vedovato and Gadsby, 2010) in the same oocyte. Inward current of (A) E336Q (n = 3) and D813N (n = 6), and of (B) E788Q (n = 7) and D817N (n = 5), pumps, like that of control C113Y pumps (n = 12–16), increased substantially on lowering pHo from 7.6 to 6.0; inward current at pHo 6.0 and −180 mV in E336Q, D813N, and D817N was >60%, and in E788Q it was 43%, that of C113Y pumps. But inward current of (C) D935N (n = 5) and E963Q (n = 4) pumps was comparatively small at pHo 6.0, and that of (D) D935N/E963Q double mutant (cyan; n = 3) pumps was almost absent compared with C113Y pumps (n = 16); as K+o-activated currents of D935N and D935N/E963Q pumps fail to reach a voltage-independent maximum at positive potentials (Figs. 9 and S2), their normalized inward current magnitude shown here is overestimated (by greater or equal to threefold; Fig. 9). (E) D935 (green sticks) and E963 (red sticks) are ∼10 à apart in the Xenopus α1 Na+/K+ pump homology model of the E2 · MgF42− conformation (Fig. 3 B), with the hydroxyl of TM5 Y780 (purple sticks) between them; view magnified 2.5 times, from the right-hand image of Fig. 3 B. (F) The response of inward current in Y780F (n = 4) mutant pumps to lowering pHo from 7.6 to 6.0 is small, as in D935N or E963Q pumps, compared with that of C113Y pumps.
	Figure 5. Closing the cytoplasmic Na+ and K+ access pathway in C113Y pumps by using BeFX to restrict them to phosphorylated-like states does not impair inward proton current. (A) Outward current in C113Y Na+/K+ pumps at −20 mV in Na+o-free solution was twice activated by 15 mM K+o before online injection of 1 mM BeFX into the oocyte (black downward arrow and gray current recording). The slow current decay in 15 mM K+o after BeFX injection reflects accumulation of pumps in stable E2P-like states incapable of Na+/K+ exchange (Olesen et al., 2007; Toyoshima et al., 2007). The numbers mark the voltage trials used to obtain the subtracted currents shown in B and C. (B and C) BeFX abolished ouabain-sensitive outward current in 15 mM K+o at positive voltages (B; open vs. closed triangles) but did not alter inward current in 0 K+o (C; open vs. closed circles). BeFX also prevented K+o inhibition of inward current at negative voltages (B; open vs. closed triangles); two other identical experiments gave the same results, as did nine experiments with C113Y-ΔYY pumps (C-terminal truncation mutant; Vedovato and Gadsby, 2010) in 0 Na+o (n = 6) or in 125 mM Na+o (n = 3).
	Figure 6. BeFX-bound Na+/K+ pumps mimic conformational transitions of normal phosphorylated pumps. (A and B) C113Y Na+/K+ pump–mediated pre–steady-state Na+ charge movements (e.g., Nakao and Gadsby, 1986; Meier et al., 2010; Vedovato and Gadsby, 2010) elicited by steps from −20 mV to potentials between −180 and +60 mV, in 125 mM Na+o, 0 K+o before (A) and after (B) oocyte injection with 1 mM BeFX (as in Fig. 5 A). (C) Ouabain-sensitive charge, ΔQ, on termination of each step is plotted against step voltage, and ΔQ-V is fitted with the Boltzmann relation to determine total charge moved, Qtot, effective valence, zq, and midpoint voltage; none was altered by BeFX injection. The ratio of Qtot before versus after BeFX was 1.2 for A and B, and averaged 1.1 ± 0.1 (n = 3). The same results were obtained with C113Y-ΔYY mutants (Qtot ratio = 1.15 ± 0.02; n = 4). In contrast, the ratio of K+o-activated outward pump current after versus before BeFX injection was ≤0.2 for C113Y pumps (e.g., outward current was reduced by 88% for the oocyte of A and B, and by ∼90% for C113Y-ΔYY pumps). (D) Mean (n = 3) relaxation rates of the transient currents elicited by the ON voltage steps, plotted against voltage, were not altered by BeFX. The unaltered transient Na+ charge movements demonstrate that BeFX-bound and inhibited Na+/K+ pumps remain capable of a subset of normal conformational changes in 125-mM Na+o solutions.
	Figure 7. Preventing extracellular access to the principal Na+ and K+ transport pathway does not impair inward proton current in C113Y-ΔYY Na+/K+ pumps. (A) Recording of abolition by 1 mM MTSET+ of K+o-activated outward current in T806C(C113Y-ΔYY) mutant Na+/K+ pumps, at −50 mV in 125-mM Na+o solutions; the ΔYY mutant was chosen as background because that C-terminal truncation amplifies proton currents in 125 mM Na+o (Yaragatupalli et al., 2009; Poulsen et al., 2010; Vedovato and Gadsby, 2010), the condition in which MTSET+ abolished large Na+ currents in palytoxin-bound Na+/K+ pump channels (Reyes and Gadsby, 2006; Takeuchi et al., 2008). 10 mM dithiothreitol (bar labeled “DTT”) was first applied to reverse any spontaneous cysteine oxidation. Outward pump current was assessed before and after the modification by MTSET+ (bar labeled “MTSET+”). Black and red stars, respectively, mark voltage trials that typically yielded the average subtracted currents shown in B (at 15 mM K+o) and C (at 0 K+o). (B) Ouabain-sensitive currents were normalized to the outward pump current between 0 and +60 mV in 15 mM K+o before MTSET+ in each oocyte, and then averaged. In 15 mM K+o, outward current at positive voltages was reduced by 90 ± 0.02% (n = 7 oocytes) by MTSET+, but current was inward at large negative potentials, indicating that K+o could no longer inhibit inward proton current. (C) Inward currents in 0 K+o were normalized to the ouabain-sensitive outward current between 0 and +60 mV in 15 mM K+o before MTSET+ in each oocyte, and then averaged. MTSET+ modification of T806C did not diminish inward current amplitude at −180 mV (n = 7 oocytes), although voltage sensitivity was altered.
	Figure 8. Wild-type Na+/K+ pump currents in 125 mM Na+o at normal and lowered pHo. (A) Current record at −20 mV (same oocyte as in Fig. 2, A and B) during the addition of 15 mM K+o and ouabain, as indicated, at pHo 6.0 (gray current trace) and pHo 7.6 (black trace). (B) Ouabain-sensitive steady currents without (squares) and with (stars) 15 mM K+o, at pHo 7.6 (closed symbols) and 6.0 (open symbols), from subtraction of currents in numbered voltage trials in A (the same results obtained in n = 8 oocytes are shown averaged in Fig. S4 A).
	Figure 9. Comparing expression and function of D935N(C113Y) and parent C113Y pumps. (A and B) Representative recordings of pump–channel currents induced by 100 nM palytoxin (PTX), at −50 mV with symmetrical 125-mM [Na+] solutions, in outside-out patches excised from oocytes injected with the same amounts of cRNA (30 ng α subunit plus 10 ng β subunit): (A) after 1 d of expression (black, C113Y: steady current amplitude, −1.2 ± 0.2 nA and n = 4; green, D935N: −1.1 ± 0.2 nA and n = 4), and (B) after 2 d (black, C113Y: −2.1 ± 0.2 nA and n = 15; green, D935N: −2.1 ± 0.1 nA and n = 8). (C) Ouabain-sensitive outward Na+/K+ transport currents at 15 mM K+o in 125 mM Na+o from the very same two C113Y-expressing (black stars) and D35N-expressing (green stars) oocytes from which the patch recordings in B were obtained. (D) Average outward pump currents in 15 mM K+o and 125 mM Na+o for C113Y (black stars; n = 12) and D935N (green stars; n = 9) after 2 d of expression, recorded in oocytes (including those of B and C) from the same batches that yielded the average excised-patch data in B.

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