Article pubs.acs.org/biochemistry Role of the Juxtamembrane Region of Cytoplasmic Loop 3 in the Gating and Conductance of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Yassine El Hiani and Paul Linsdell* Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada *S Supporting Information ABSTRACT: Opening and closing of the cystic ﬁbrosis transmembrane conductance regulator chloride channel are controlled by interactions of ATP with its cytoplasmic nucleotide binding domains (NBDs). The NBDs are connected to the transmembrane pore via four cytoplasmic loops. These loops have been suggested to play roles both in channel gating and in forming a cytoplasmic extension of the channel pore. To investigate the structure and function of one of these cytoplasmic loops, we have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced into loop 3. We ﬁnd that methanethiosulfonate (MTS) reagents modify cysteines introduced at 14 of 16 sites studied in the juxtamembrane region of loop 3, in all cases leading to inhibition of channel function. In most cases, both the functional eﬀects of modiﬁcation and the rate of modiﬁcation were similar for negatively and positively charged MTS reagents. Single-channel recordings indicated that, at all sites, inhibition was the result of an MTS reagent-induced decrease in channel open probability; in no case was the Cl− conductance of open channels altered by modiﬁcation. These results indicate that loop 3 is readily accessible to the cytoplasm and support the involvement of this region in the control of channel gating. 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. We propose that either the cytoplasmic pore is very wide or cytoplasmic Cl− ions use other routes to access the transmembrane pore. C ystic cystic ﬁbrosis ﬁbrosis is caused transmembrane by loss of function conductance mutations regulator in the (CFTR) Cl− channel, a member of the ATP-binding cassette (ABC) family of ATP-dependent membrane transport proteins. All ABC proteins share a common modular architecture, consisting of two membrane-spanning domains (MSDs) that form the substrate translocation pathway and two cytoplasmic nucleotide binding domains (NBDs) that bind and hydrolyze ATP (Figure 1). In CFTR, an additional cytoplasmic regulatory domain (R domain) is the site of regulation by PKA-dependent phosphorylation. Consistent with this ABC architecture, several transmembrane (TM) α-helices1−3 and extracellular loops (ELs)4,5 have been shown to contribute to the Cl− channel pore in CFTR. The activity of the channel is controlled by ATP interactions at the NBDs,6,7 which leads to the opening and closing of a “gate” located in the MSDs.8 The NBDs are not in direct contact with the TMs but instead are connected indirectly via the long cytoplasmic loops (CLs) that are located between individual TMs (Figure 1). Structural models of the CFTR protein9−11 therefore suggest that NBD− CL interactions should be important in coupling ATP action at the NBDs to channel opening in the MSDs. In fact, the role of the CLs in forming a physical and functional link between the NBDs and the transmembrane substrate translocation pathway may be conserved among all ABC proteins.12,13 The location of the CLs below the TMs (Figure 1A) also suggests that the CLs could form a cytoplasmic extension of the permeation pathway for Cl− ions. In fact, on the basis of these models, it has been suggested that the CLs form a narrow “funnel” connecting the TMs to the cytoplasm (Figure 1C), and that this CL funnel, not the TMs, forms the narrowest part of the Cl− channel pore where channel opening and closing may occur.11 Functional evidence also supports a role for NBD−CL interactions in ATP-dependent channel gating (pore opening and closing). For example, CL3, located between TM8 and TM9 (Figure 1), interacts with both NBDs14 as well as the R domain15,16 to regulate gating. Mutations within CL3 also aﬀect gating,17,18 perhaps by disrupting communication between the NBDs and the gate in the MSDs.19 CF-associated mutations in this loop also disrupt processing and traﬃcking of CFTR protein to the membrane,17,20 perhaps highlighting the importance of domain−domain interactions in proper protein folding,10,21−23 although CL3 has also been implicated in ubiquitination-dependent CFTR traﬃcking.24 There is also some functional evidence that CL3 may contribute to the Cl− permeation pathway. The CF-associated mutations S945L and G970R were shown to have very slightly altered single-channel Cl− conductance.17 Furthermore, the positive charge associated Received: January 16, 2012 Revised: April 30, 2012 Published: April 30, 2012 © 2012 American Chemical Society 3971 dx.doi.org/10.1021/bi300065z | Biochemistry 2012, 51, 3971−3981