Biochemistry Article Figure 1. Location of CL3 within the overall structure of CFTR. The CFTR protein consists of two MSDs and two NBDs, joined by a cytoplasmic R domain. Each MSD consists of six TM helices connected by three ELs and two CLs. (A) Proposed overall structure of CFTR (viewed from the side), based on atomic homology modeling of bacterial ABC protein Sav1866.10 TMs that are known to line the Cl− channel pore are colored red (from left to right, TM1, TM6, and TM12, respectively). The locations of TM8 (left) and TM9 (right) are colored blue. Connecting these two TMs is CL3, which is colored blue, with the region mutated in this study colored gold. (B) Isolated TM8−CL3−TM9 region from the same model, showing the juxtamembrane region of this CL as a cytoplasmic extension of these two TMs. (C) Position of the CLs around a narrow “funnel” region in the cytoplasm, as viewed from above. Other parts of the protein have been removed for the sake of clarity. The part of CL3 that appears to line this narrow region is that proximal to TM9. Illustrations were prepared using PyMOL based on the structure presented by Serohijos et al.10 (D) Two-dimensional topology of CFTR, illustrating two MSDs, two NBDs, the R domain (RD), and 12 TMs. The coloring of the TMs is the same as that in panel A. The region mutated in this study is enclosed in the dotted box, which is magniﬁed in panel E. This shows the amino acid sequence of CL3, from P936 at the cytoplasmic end of TM8 to P988 at the cytoplasmic end of TM9. As in panels A−C, residues mutated in this study (L941− K951 and L973−L983) are colored gold. with CL3 residue K978 is involved in interactions with cytoplasmic channel blocking substances.25,26 However, the role of CL3 in channel function has not previously been addressed in a comprehensive way. To investigate the possibility that CL3 forms a cytoplasmic extension of the CFTR pore, we have undertaken a substituted cysteine accessibility mutagenesis (SCAM) study of the juxtamembrane parts of this loop. Because residues within the CLs have not previously been directly demonstrated to contribute to the permeation pathway, we reasoned that this region relatively close to the TMs might be considered most likely to inﬂuence Cl− permeation. Furthermore, more cytoplasmic parts of CL3 are likely to be involved in interactions with NBD1.9,10,14 We have used SCAM because of the proven power of this approach in identifying amino acids that contribute to the CFTR permeation pathway.2,3,27−30 Our results indicate that the juxtamembrane regions of CL3 are exposed to the cytoplasm and are readily modiﬁed by intracellular cysteine-reactive methanethiosulfonate (MTS) reagents. However, the eﬀects of modiﬁcation appear to reﬂect changes in channel gating, rather than channel permeation properties. These results suggest that while CL3 plays a role in the coordination of ATP-dependent gating, it does not make an important functional contribution to the Cl− permeation pathway. ■ MATERIALS AND METHODS Experiments were conducted on baby hamster kidney (BHK) cells transiently transfected with CFTR. As in our recent SCAM-based studies of individual TMs,2,28,30 we have used a human CFTR variant from which all cysteines had been removed by mutagenesis (as described in ref 31) and which includes a mutation in NBD1 (V510A) to increase the level of protein expression in the cell membrane.32 Use of this Cys-less variant is necessary for these studies because wild-type CFTR is potently inhibited by cytoplasmic MTS reagents.33 However, in 3972 dx.doi.org/10.1021/bi300065z | Biochemistry 2012, 51, 3971−3981