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.