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Figure 3. Forward pumping currents of WT and p.S779N α 2 at various extracellular [K+]o. (A) Example current traces of wild-type (black) and mutant (red) pumps in response to 200 ms steps from −160 to +40 mV, from a holding voltage of −30 mV in absence (left) and presence (middle) of 15 mM K+, and in presence of 15 mM K+ and 10 mM ouabain (right). K+-induced pump currents were isolated off-line by subtraction of currents in the presence of ouabain 10 mM. (B) Current-voltage relationship of steady state ouabain-sensitive currents measured in last 50 ms of each step (p.S779N n = 17; wild-type n = 20; P < 0.0001 two-way ANOVA). (C) Current-voltage relationship of ouabain-sensitive currents normalized to total Na+ charge transfer (Qtot) in absence of K+, as a measure of functional protein expression (p.S779N n = 7; wild-type n = 13; P = 0.001, two-way ANOVA). Qtot was estimated in a subset of cells where lower amplifier gain was used to minimize current saturation because of capacitive artefacts. Ten traces were averaged to reduce the noise and then used to determine the span of the Boltzman curve (Qtot: S779N 5.4 ± 0.9 nC, n = 7; wild-type 5.5 ± 0.7 nC, n = 13). (D) Extracellular apparent K+ affinity of the ATPases: current-voltage relationships of wild-type (i) and mutant pumps (ii) in various [K+]o conditions normalized to the outward current at +20 mV in 15 mM K+ (p.S779N n = 5–6; wild-type n = 3–5). (E) Concentration-response curves obtained from the data in D at three representative voltages. Dotted lines represent fits to a modified Hill equation (wild-type EC50 at −100, −60, 0 mV: 3.5 ± 0.3, 2.2 ± 0.2, 1.3 ± 0.1 mM, n = 5; P < 0.0001 repeated measures ANOVA; p.S779N EC50 at −100, −60, 0 mV: 6.9 ± 0.4, 6.9 ± 0.2, 7.5 ± 0.7 mM, n = 5; P = 0.7). Curves obtained from separate experiments were normalized to the top and bottom values of the fit and averaged across oocytes. Hill coefficients were voltage independent and did not change significantly between pump variants: wild-type 1.54 ± 0.06, S779N 1.56 ± 0.06 (P = 0.3, two-way ANOVA across the voltages). (F) Overall voltage dependence of apparent [K+] affinity (wild-type n = 3–5; p.S779N n = –5, P < 0.001 two-way ANOVA). Image published in: Sampedro Castañeda M et al. (2018) © The Author(s) (2018). This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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