FIGURE 6.
Mutant vGAAPs have altered single-channel properties. Spontaneous single-channel currents produced by purified WT, E207Q, and D219N CMLV GAAPs in planar lipid bilayers were measured during stepwise changes in membrane potential (A–D) or, for WT and E207Q GAAPs, from repetitive voltage ramps from −150 mV to +150 mV over 1 s (E and F). A, comparison of single-channel currents produced by WT and mutant GAAPs at −40 mV (n > 4 independent bilayers). Representative traces are shown in B, with the closed state indicated by a dotted line. C, current-voltage (i-V) relationships for single-channel currents measured at different voltages (n = 4–6 independent bilayers). D, single-channel open probability (Po) measured at −40 mV from 4–7 bilayers for WT and mutant CMLV GAAP proteins. Results show means ± S.E. (error bars) (**, p < 0.01). E, representative recordings of WT CMLV GAAP and the E207Q mutant channel activity during voltage ramps. The closed state of the channel is indicated by a dotted line. F, the current-voltage relationships of single-channel currents produced by WT CMLV GAAP and mutant E207Q measured from voltage ramp recordings (n > 14 independent bilayer recordings). G, single-channel conductances calculated from continuous and voltage ramp recordings (n = 4–5 and n > 14 independent bilayers, respectively). Conductance measurements for the D219N mutant were restricted to negative voltages. H, reversal potentials, measured from single-channel currents recorded during stepwise changes in voltage or voltage ramps (n = 3–5 and n > 8 independent bilayers). A, D, G, and H, statistical analyses relative to WT GAAP were made using an unpaired Student's t test (A) or one-way analysis of variance (D, G, and H) followed by Dunnett's (D) or Newman-Keuls multiple comparison tests (G and H); data shown as means ± S.E. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).