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. 2022 Jun 1;11:e77030. doi: 10.7554/eLife.77030

Figure 4. Accessibility of residue A193C supports voltage-dependent motion of S4 segment.

Figure 4.

(A) Cartoon representing extracellular cysteine accessibility of residue A193C as in Figure 1A. (B) Currents in response to +20 mV voltage steps before (gray trace #0) and during several 5 s applications of methanethiosulfonate (MTSET) at +20 mV (green traces #1–7), –100 mV (blue traces #1–12), and –140 mV (black traces #1–19) on A193C channels for the indicated voltage protocol. We repeat MTSET applications (10 μM at +20 and –100 mV, and 20 μM at –140 mV) in between 25 s washouts as shown in each voltage protocol. (C) Normalized current of A193C during MTSET exposure at +20 mV (green), 0 mV (orange), – 80 mV (pink), –100 mV (blue), and – 140 mV (black). (D and E) Normalized G(V) curves (squares and black line from a Boltzmann fit) of A193C channels and voltage dependence of the modification rate (mod. rate [V], green circles and green line from a Boltzmann fit) for MTSET to residue A193C. In (E), dashed lines represent ‘wt’ KCNQ2* (black) G(V) and (cyan) F(V) curves for comparison. (E’) Summary of G(V)1/2 for (open squares) A193C and (open gray circles) labeled F192C-Alexa channels. Data are presented as mean ± SEM, n=9–12. Statistical significance was determined using paired Student t-test and significance level was set at p<0.05, p=0.062. The midpoints of activation for the fits are: GV1/2A193C = – 70 ± 2.4 mV, (n=12) and GV1/2F192C-Alexa = –77.1 ± 2.7 mV, (n=9); Mod. rate V1/2 A193C = –72.8 ± 24.5 mV, (n=3–8); GV1/2A193C = – 70 ± 2.4 mV, (n=12); see Figure 2G for KCNQ2* GV1/2 and FV1/2, values.