Closing the cytoplasmic Na⁺ and K⁺ access pathway in C113Y pumps by using BeF_X 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 BeF_X into the oocyte (black downward arrow and gray current recording). The slow current decay in 15 mM K⁺_o after BeF_X 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) BeF_X 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). BeF_X 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).