![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).](https://cdn.rupress.org/rup/content_public/journal/jgp/154/7/10.1085_jgp.202113039/3/jgp_202113039_fig4.png?Expires=1767710029&Signature=vKoAeJx-fu4Rb-nuz8f~-7gkxWjMbO8NuHoGKj3Tn5KDKn~hnyLuTD6-C0cQcFM3Lv-YwlJMApuR2mhhype-B7iZMSdfgiQxXVBNYQ~Ea8~JJu3LTy26BdIfw4EqaD~FY8CWOUxGa~gdxbeaB8rb2DltBjjx~RxZdMuBmdxtlfl1Q4~wTKQJsREcgQSn~XfOhzrBNuYsCvo6cRezQRd7-au05EhPg55bFEfVipx1vdDXwZDEdTta1StGET7PUPrSAJJAKr0vNAuUXUrvpXPJueHk~IqgsDmn~WDUX6uJdR3WKXBkLFtQeEw8M3MTP64c3wz9att358-s5TlVby4dFg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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).