Biochemistry Article Figure 6. Single-channel conductance of cysteine mutants. (A) Example single-channel currents carried by Cys-less CFTR and the charge- substituting mutants K946C and R975C as indicated, at a membrane potential of −50 mV. The horizontal line to the left of the traces indicates the channel closed state current level. (B and C) Mean single-channel I−V relationships recorded under these conditions for Cys-less (○), K946C (●, B), and R975C (●, C). (D) Mean single-channel conductance for diﬀerent channel variants measured under these conditions. None of these were signiﬁcantly diﬀerent from the Cys-less background. Means of data from three to twelve patches are shown in panels C and D. (see below). Results with this mutant, which was shown to generate constitutively active (ATP-independent) channel, formed a central part of the hypothesis put forward by Wang et al.18 concerning the role of CL3 in channel gating. It was a great surprise to us, therefore, that we were unable to observe functional expression of K978C in our experiments (Figure 3), especially as Wang et al.18 had also studied this mutant in a Cys-less background. Because we were so surprised at not being able to replicate the functional expression of this mutant, we in fact constructed this mutant on three independent occasions, but in each case, the lack of fuctional expression remained the same. We can only imagine that some diﬀerence in the expression system has resulted in our failure to observe currents for this mutant. Indeed, our results indicate that this mutant is associated with a severe protein processing defect in BHK cells (Figure 2). In fact, the functional results of Wang et al.18 for K978C, that the open probability of the channel is reduced following modiﬁcation by MTSET, are quite consistent with our own ﬁndings for other, nearby CL3 residues. Our focus in this work was to explore the functional properties of CL3 mutants that do express functional channels in the membrane of BHK cells. We found that 14 of 16 functional cysteine-substituted mutants were sensitive to cytoplasmic exposure to MTSES and/or MTSET (Figure 4). This suggests that most of the juxtamembrane parts of CL3 (Figure 1) are exposed to the cytoplasm and readily accessible to large cytoplasmic substances. In fact, there was no apparent diﬀerence between those mutations located proximal to TM8 compared to those proximal to TM9, in terms of the functional eﬀect of modiﬁcation (Figure 4) or the rate of modiﬁcation (Figure 5), suggesting that these two “limbs” of CL3 show approximately equivalent accessibilities to the cytoplasm. We 3977 dx.doi.org/10.1021/bi300065z | Biochemistry 2012, 51, 3971−3981