Fig. 3: Functional and structural characterization of WT-, K794S-, and K794A-ngHKA.

a Representative results from more than three independent measurements of K⁺-dependent ATPase in crude membrane fractions from cells expressing ngHKA WT (blue), K794A (green) and K794S (yellow), measured in the absence (open symbols) or in the presence (filled symbols) of 100 mM Na⁺. The maximum K⁺-activated activity without Na⁺ was set as 100% and that without K⁺ or Na ⁺, as blank. Line plots are Hill fits (see “Methods”). b Current at −50 mV from Na⁺-loaded oocytes expressing WT-, K794A-, and K794S-ngHKA or NKA pumps. Application of K⁺ activated outward currents only in oocytes expressing K794A-ngHKA (K_0.5 = 2.8 ± 0.7 mM, nH = 1.75, n =  5) or NKA (K_0.5 = 1.21 ± 0.34 mM, nH = 1.47, n = 9). Note similar K_0.5 for current and ATPase activation at 100 mM Na⁺ for Lys794Ala. Ouabain (25 mM ngHKA & 10 mM NKA) blocks subsequent responses to K⁺. c Ouabain-sensitive transient currents elicited by the pulse protocol shown on top. The protocol was repeated before and after the application of ouabain (10 mM for NKA, 25 mM for ngHKA mutants) to obtain the pump-specific signals displayed (current without ouabain minus current in ouabain). d Mean Q–V from experiments like those in (c). The Boltzmann fitted to individual experiments had V_1/2 = −75 ±  2 mV and kT/z_qe = 64 ± 3 mV (n = 16) for Lys794Ser (yellow line), and V_1/2 = −51 ± 1 mV and kT/z_qe = 40 ± 1 (n = 12) for NKA. Data for Lys794Ala (n = 7) and WT (n = 4) could not be fitted. e Ion-binding sites of K794A-ngHKA with a K⁺ ion modeled at site II. Water was modeled in the weak density near site I (side-chain oxygens of Asn795 (2.30 Å) and Asp823 (2.39 Å) are too close to accommodate K⁺). f Ion-binding sites of K794S-ngHKA. Strong densities are consistent with K⁺ ions bound at both sites. Both K⁺-occluded structures are viewed from the cytoplasmic side.