Figure 5.
γ-11 cross-links to sites in the γ subunit finger and thumb domains. Oocytes were injected with 1 ng/subunit of cRNA encoding human α lacking furin cleavage sites (αF), WT β, and γ subunits lacking the autoinhibitory tract (γΔI; control) with cysteine mutations as indicated. Sites tested were γS220C in the finger domain (A and B) and γQ426C in the thumb domain (C and D). A and C, currents were measured by two-electrode voltage clamp at −60 mV. Channels were labeled by perfusing oocytes with 10 μm MTS-4-MTS for 15 s, followed by a 30-s wash (−). Channels were then inhibited by the γ-11 cysteine derivative indicated for 60 s, followed by a wash step (−) to assess reversibility of peptide inhibition. Baseline currents were −1.0 ± 3.3 μA for γS220C, and −3.6 ± 0.6 μA for γQ426C. B and D, reversibility of peptide inhibition was measured by comparing the currents at the end of the final wash step to the currents just before peptide addition and the currents at the end of peptide addition (see schematic in A and C). Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.