Biochemistry Article Figure 8. Eﬀects of MTSES on the single-channel current amplitude and open probability. Single-channel recordings such as those shown in Figure 7 were used to quantify the eﬀect of MTSES (200 μM) on the single-channel current amplitude (A) and open probability (B) for each of the channel variants named. Means of data from three to nine patches are shown. Asterisks indicate a signiﬁcant diﬀerence from Cys-less (p < 0.05). based on homology modeling, that the CLs form a physically narrow region of the pore (Figure 1).11 It is also possible that other CLs make a more important functional contribution than CL3 does, although the model shown in Figure 1 suggests a fairly symmetrical contribution of all four CLs to the apparent central pore. An alternative possibility is that this central pore apparently formed by the CLs (Figure 1C) is not part of the Cl− permeation pathway at all. In some ion channel types, the permeant ion does not follow a direct pathway along the central axis of the entire protein but instead enters and exits a central transmembrane pore via lateral “portals” located close to the cytoplasmic side of the membrane.44−48 There is no functional evidence of the existence of such portals in CFTR, although we would point out that there is also no direct evidence that Cl− ions enter the permeation pathway at the cytoplasmic end of the CLs and pass along the extent of the central axis of the channel protein. Interestingly, a recent direct structural study of CFTR suggested that portals might exist at the TM5−CL2 and TM11−CL4 cytoplasmic boundaries.49 In many ABC proteins, the substrate translocation pathway is partially open to part of the lipid bilayer, raising the possibility that some substrates enter and exit via the bilayer.50 As described by Bai et al.,29 such an opening to the bilayer, which can actually be observed at the extracellular end of the CFTR structural model of Figure 1A, is inconsistent with the function of CFTR as an ion channel. Nevertheless, these openings do raise the precedent that ABC protein substrates do not necessarily follow an unwaveringly central path through the protein. Our results do support the previously suggested role for CL3 in gating of the CFTR channel.14−18 MTSES modiﬁcation of 14 of 16 functional cysteine-substituted mutants led to a decrease in channel open probability (Figure 8B), even though the mutations themselves did not appear to have dramatic eﬀects on open probability (Figure S2 of the Supporting Information). In most cases, MTSET had a similar eﬀect (Figure 4), indicating a lack of strong charge dependence in the functional eﬀects of modiﬁcation. The consistent eﬀects of MTS modiﬁcation (Figure 4) argue against a speciﬁc role for individual amino acid side chains in CL3 in channel gating and instead suggest that changes in the structure of this CL exert a relatively nonspeciﬁc eﬀect on gating. This would appear to be consistent with the hypothesis put forward by Wang et al.18 that CL3 forms a “compression spring” that regulates the opening and closing of the channel. Modiﬁcation by MTS reagents may then stiﬀen the spring and reduce the open probability in a relatively position-independent fashion. This hypothesis emphasizes the role of the CLs in transmitting gating information “forward” from the NBDs to the pore. It is also possible that the CLs are involved in transmitting gating information “backward”, i.e., that changes in the structure of CL3 feed back to modify NBD function, ultimately resulting in a change in channel gating.29,51 Irrespective of the precise mechanism, our similar results for mutations in the two limbs of 3979 dx.doi.org/10.1021/bi300065z | Biochemistry 2012, 51, 3971−3981