FIGURE 4.
Modification of introduced cysteines during pretreatment with internal MTSES. A–C, example leak-subtracted I-V relationships for five highly MTSES-sensitive mutants (T338C, S341C, I334C, V345C, M348C) showing the effects of application of internal MTSES (200 μm) after maximal channel activation with PKA (20 nm), ATP (1 mm), and PPi (2 mm). Patches have been pretreated in three different ways (see “Experimental Procedures”), no pretreatment (A), pretreated with MTSES (200 μm), PKA, and ATP for 2 min (B), and pretreated with MTSES alone (200 μm) for 2 min (C). Note that for each mutant, after pretreatment with MTSES, PKA, and ATP, stimulated currents were small and apparently refractory to the effects of internally applied MTSES (B), suggesting that channels had been covalently modified during the pretreatment. D, shown is the mean effect of internal MTSES on macroscopic current amplitude at −80 mV in each of the three protocols shown in A–C. Asterisks indicate a significant difference from control (no pretreatment, A) conditions (p < 0.01). Data are the means from 3–4 patches.