Fig. 2.

Modification of cysteine-substituted TM8 mutants by extracellular MTS reagents. a Examples of the time course of whole-cell currents carried by Y914C, Y917C and Y919C-CFTR (cys-less background). As described in “Materials and methods”, current amplitude was monitored using voltage ramps (see b) and is plotted at membrane potentials of − 50 mV (open circle) and + 50 mV (closed circle). Currents were activated by extracellular application of cAMP stimulatory cocktail (see “Materials and methods”) as indicated by the gray bar marked cAMP. After activation, cells were treated with negatively charged MTSES⁻ (1 mM; red bar for Y914C and Y919C) or positively charged MTSET⁺ (1 mM; blue bar for Y917C). At the end of the experiment, current was confirmed as CFTR-mediated using GlyH-101 (20 μM; black bar marked GlyH). b Example current (I)–voltage (V) relationships for these cells, recorded during voltage ramps before (control; black lines) and after (red lines for MTSES⁻, blue line for MTSET⁺) exposure to MTS reagent. Note the inward rectification of the control I–V curves for Y914C and Y917C. c Mean fraction of control current remaining following exposure to MTSES⁻ (red bars) or MTSET⁺ (blue bars) for these and other TM8 cysteine-mutant channels. The effects of MTS reagents were not appreciably voltage-dependent, and the effects of these reagents were averaged across the voltage range used. Asterisks indicate a significant difference from control, pre-MTS current levels (p < 0.05). Mean of data from 3–4 cells