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. 2005 Sep;126(3):213–226. doi: 10.1085/jgp.200509287

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Copyright © 2005, The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure 3. — Functionality tested in Gly mutants. (A) Functional Gly scan using FLAG-tagged double mutants expressed in Xenopus oocytes. Peak currents at +50 mV. All mutants contained G466A and cRNAs were injected at equimolar concentrations. Recordings were obtained 2 d after injection, n = 3–6 eggs. Similar results were obtained for all double mutants using mammalian cell expression (not depicted). (B) Cs⁺ currents obtained in whole cell recording as described in Fig. 1 A. (C) G-V relationships for Cs⁺ currents in WT, V467G, and G466A/V467G constructs (n = 4 in each case) in mammalian cells. These relationships were determined from isochronal tail current measurements. The G-V relations were fitted to the Boltzmann equation:

G V=,

where G(V) is normalized conductance, V _1/2 is the half activation voltage, q is the unitless slope, and RT/F is 25 mV at room temperature. For WT: V _1/2, q was −40.5 ± 0.8 mV, 4.99 ± 0.13; for V467G: V _1/2, q was −55.2 ± 2.4 mV, 3.50 ± 0.23; for G466A/V467G: V _1/2, q was −22.0 ± 1.2 mV, 2.16 ± 0.11. Free energy differences were calculated as ΔΔG = Δ(qFV _1/2) for any two constructs. Although this is only a crude measure of the open–closed equilibrium energy, an improved approach based on the assumption that S6 mutants affect only the concerted opening transition (Yifrach and MacKinnon, 2002) is inconsistent with the differences between WT and the V467G mutant, because the mutation causes the G-V relationship to be both left shifted and shallower than that of the WT.

Inline graphic — Functionality tested in Gly mutants. (A) Functional Gly scan using FLAG-tagged double mutants expressed in Xenopus oocytes. Peak currents at +50 mV. All mutants contained G466A and cRNAs were injected at equimolar concentrations. Recordings were obtained 2 d after injection, n = 3–6 eggs. Similar results were obtained for all double mutants using mammalian cell expression (not depicted). (B) Cs⁺ currents obtained in whole cell recording as described in Fig. 1 A. (C) G-V relationships for Cs⁺ currents in WT, V467G, and G466A/V467G constructs (n = 4 in each case) in mammalian cells. These relationships were determined from isochronal tail current measurements. The G-V relations were fitted to the Boltzmann equation:

G V=,

where G(V) is normalized conductance, V _1/2 is the half activation voltage, q is the unitless slope, and RT/F is 25 mV at room temperature. For WT: V _1/2, q was −40.5 ± 0.8 mV, 4.99 ± 0.13; for V467G: V _1/2, q was −55.2 ± 2.4 mV, 3.50 ± 0.23; for G466A/V467G: V _1/2, q was −22.0 ± 1.2 mV, 2.16 ± 0.11. Free energy differences were calculated as ΔΔG = Δ(qFV _1/2) for any two constructs. Although this is only a crude measure of the open–closed equilibrium energy, an improved approach based on the assumption that S6 mutants affect only the concerted opening transition (Yifrach and MacKinnon, 2002) is inconsistent with the differences between WT and the V467G mutant, because the mutation causes the G-V relationship to be both left shifted and shallower than that of the WT.