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 pH_o (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, Q_tot, 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 pH_o from 7.6 to 6.0; inward current at pH_o 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 pH_o 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 Å 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 pH_o from 7.6 to 6.0 is small, as in D935N or E963Q pumps, compared with that of C113Y pumps.