Biochemistry CL3 suggest these regions of the protein play similar functional roles. Furthermore, because MTS modiﬁcation of most residues resulted in a similar, ∼25−75% inhibition of the macroscopic current amplitude, it might be considered that no sites in CL3 are absolutely essential to channel function, perhaps pointing to a role of this region as a mediator, rather than a controller, of channel gating. In summary, our comprehensive functional screening of CL3 suggests that this region of CFTR is readily accessible to the cytoplasm, where it plays a role in the control of channel gating. This is consistent with the prevailing idea that CL3 forms the link between the site of ATP action (the NBDs) and the opening and closing of a channel gate located in the TMs. However, our results do not support the hypothesis that this region is close enough to the Cl− permeation pathway to exert any inﬂuence on permeating Cl− ions. ASSOCIATED CONTENT *S Supporting Information Two ﬁgures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. ■ ■ ■ ■ AUTHOR INFORMATION Corresponding Author *Phone: (902) 494-2265. Fax: (902) 494-1685. E-mail: paul. linsdell@dal.ca. Funding This work was supported by the Canadian Institutes of Health Research. Y.E.H.is a Cystic Fibrosis Canada Postdoctoral Fellow. Notes The authors declare no competing ﬁnancial interest. ABBREVIATIONS ABC, ATP-binding cassette; BHK, baby hamster kidney; CFTR, cystic ﬁbrosis transmembrane conductance regulator; CL, cytoplasmic loop; EL, extracellular loop; MSD, membrane- spanning domain; MTS, methanethiosulfonate; MTSES, [2- sulfonat oethyl] met haneth iosulfonate; MTSET, [2- (trimethylammonium)ethyl] methanethiosulfonate; NBD, nu- cleotide binding domain; PKA, protein kinase A; SCAM, substituted cysteine accessibility mutagenesis; TM, trans- membrane α-helix. REFERENCES (1) Linsdell, P. 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