Figure 5. Modification of introduced... | Rockefeller University Press

Figure 5.

$Figure 5. Modification of introduced cysteines during pretreatment with internal MTSES. (A and B) Example leak-subtracted I-V relationships for each of the four MTSES-sensitive mutants named, showing the effects of the application of internal MTSES (200 µM) after maximal channel activation with 20 nM PKA, 1 mM ATP, and 2 mM PPi. Patches have been pretreated in two different ways (see Materials and methods): (A) pretreated with 200 µM MTSES, PKA, and ATP for 2 min; (B) pretreated with 200 µM MTSES alone for 2 min. Similar examples for patches that underwent no pretreatment are shown in Fig. 1 B. Note that for each mutant, after pretreatment with MTSES, PKA, and ATP, stimulated currents appeared refractory to the effects of internally applied MTSES (A), suggesting that channels had been covalently modified during the pretreatment. (C) Mean effect of internal MTSES on macroscopic current amplitude at +80 mV under three different sets of condition as indicated: no pretreatment (see Fig. 1 B); pretreated with MTSES, PKA, and ATP (A); and pretreated with MTSES alone (B). Asterisks indicate a significant difference from control (no pretreatment) conditions (P < 0.005); other groups not marked by an asterisk showed no significant difference from control conditions (P > 0.3). Mean of data from three to six patches is shown.$

Modification of introduced cysteines during pretreatment with internal MTSES. (A and B) Example leak-subtracted I-V relationships for each of the four MTSES-sensitive mutants named, showing the effects of the application of internal MTSES (200 µM) after maximal channel activation with 20 nM PKA, 1 mM ATP, and 2 mM PPi. Patches have been pretreated in two different ways (see Materials and methods): (A) pretreated with 200 µM MTSES, PKA, and ATP for 2 min; (B) pretreated with 200 µM MTSES alone for 2 min. Similar examples for patches that underwent no pretreatment are shown in Fig. 1 B. Note that for each mutant, after pretreatment with MTSES, PKA, and ATP, stimulated currents appeared refractory to the effects of internally applied MTSES (A), suggesting that channels had been covalently modified during the pretreatment. (C) Mean effect of internal MTSES on macroscopic current amplitude at +80 mV under three different sets of condition as indicated: no pretreatment (see Fig. 1 B); pretreated with MTSES, PKA, and ATP (A); and pretreated with MTSES alone (B). Asterisks indicate a significant difference from control (no pretreatment) conditions (P < 0.005); other groups not marked by an asterisk showed no significant difference from control conditions (P > 0.3). Mean of data from three to six patches is shown.

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