Fig. 3.
Accessibility of S3b residues of short S3–S4 linker mutants. (A) Effects on the voltage-dependent activation in the S3 cysteine mutants with short-linker (LAI) background. •, LAI (control); ○, I315C; ▴, I321C; ♦, F324C; +, T326C; □, A328C. Each point is the average of determinations on four to seven separate patches. Solid lines were drawn by using Eq. 1. V1/2 values were –15 ± 8 mV, 7 ± 2 mV, –19 ± 2 mV, 32 ± 1 mV, 26 ± 5 mV, and 22 ± 9 mV for LAI (control), I315C, I321C, F324C, T326C, and A328C, respectively. The equivalent number of gating charges, zeq, was 2.2 ± 0.5, 1.8 ± 0.4, 4.5 ± 1.3, 0.9 ± 0.04, 1.1 ± 0.4, and 0.8 ± 0.4 for LAI (control), I315C, I321C, F324C, T326C, and A328C, respectively. (B) External accessibility of cysteines to MTSET in a short linker Shaker channel (LAI). Cysteines were in positions 328, 326, 324, and 321 in the S3b segment and in position 315 in C-terminal end of S3a. All tested positions were accessible to MTSET in both the open/closed and the closed states. Rates expressed in M–1·s–1 are given for representative experiments on each of the mutants. Note that the control LAI channel contains a cysteine in position 308 that is inaccessible to the thiol reagent. (C) State-dependent accessibility of the different cysteine mutants. Second-order rate constants are plotted for cysteine modification in the closed (•) and open (○) states. Mutant labeled F324-C10aa is a shortened linker Shaker mutant containing a S3–S4 linker of 10 aa (SSNQAMSLAI; see Fig. 1 A) and a cysteine in position 324. Mutant labeled F324C-5aa is a short linker Shaker mutant containing a S3–S4 linker of 5 aa (MSLAI; see Fig. 1 A) and a cysteine in position 324. LAC, LCI, and CAI are short linker Shaker mutants containing a S3–S4 linker of 3 aa and cysteines in the indicated positions. Each point is the mean of three or more determinations, and rates are expressed in M–1·s–1.