Biochemistry Article Figure 3. Modiﬁcation of cysteines introduced into CL3 by internal MTS reagents. (A−C) Example time courses of macrosopic currents (measured at −50 mV) carried by L941C (A), V944C (B), and L983C (C). After patch excision and recording of baseline currents, patches were treated sequentially with 20 nM PKA and 1 mM ATP (hatched bars), 200 μM MTS reagent (left panels, MTSES, black bars; right panels, MTSET, white bars), and 5 μM CFTRinh-172 (gray bars). (D and E) I−V relationships for the patches shown in panels A and B following maximal current stimulation with PKA and ATP (control), following addition of MTSES (left panels) or MTSET (right panels), and following addition of CFTRinh- 172 (+Inh). Figure 4. Eﬀects of internal MTS reagents on macroscopic current amplitudes. Mean eﬀect of treatment with 200 μM MTSES (black bars) or MTSET (white bars) on the macroscopic current amplitude measured at −50 mV, for Cys-less CFTR and each of the cysteine-substituted CL3 mutants named. Mutants that did not yield measurable macroscopic currents in inside-out patches are shown in parentheses. Means of data from three or four patches are shown. Asterisks indicate a signiﬁcant diﬀerence from the value of Cys-less (p < 0.05). constructs studied are summarized in Figure 8. In no case did MTSES exposure signiﬁcantly aﬀect single-channel current amplitude (Figure 8A). In contrast, for all mutants for which MTSES exposure reduced macroscopic current amplitudes (Figure 4), MTSES also caused a reduction in open probability (Figure 8B). MTSES did not aﬀect open probability in Cys-less, or in two mutants that were not sensitive to MTSES at the macroscopic current level [F976C and L983C (Figure 4)] (Figure 8B), suggesting that it is an MTSES-induced reduction in open probability that is responsible for the reduction in 3975 dx.doi.org/10.1021/bi300065z | Biochemistry 2012, 51, 3971−3981