The Two Halves of CFTR Form a Dual-pore Ion Channel

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) exhibits two conductance states, 9 picosiemens (pS) and 3 pS. To investigate the origin of these two distinct conductance states, we measured the single-channel activity of three truncated forms of CFTR. These include: TNR, which contains the first transmembrane domain, the first nucleotide binding domain, and the R domain; RT2N2, which contains the R domain, the second transmembrane domain, and the second nucleotide-binding domain; and T2N2, which contains only the second transmembrane domain and the second nucleotide-binding domain. The results show that TNR exhibits only the large conductance of 9.2 pS, whereas RT2N2 and T2N2 exhibit only the small conductance (3.8 -4.0 pS). Co-expression of TNR with T2N2 resulted in a mixed pattern of two conductance states, which is similar to that observed in wild-type CFTR. In further studies, a "dual-R mutant," R334W and R347P in the transmembrane segment 6 of the first half of CFTR, severely impaired the large conductance channel without affecting the small conductance channel. The ion selectivity and gating behavior of the two conductance channels are different regardless of whether they are measured in wild-type CFTR or in truncated CFTRs. The ion selectivity of the large conductance channel is Br ؊ > Cl ؊ > I ؊ , whereas the ion selectivity of the small conductance channel is BrThe open probability (P o ) of the large conductance is about 4-fold higher than that of the small conductance. Transition from closed to open states of the small conductance is not dependent upon the open or closed states of the large conductance. The independent behaviors of the two conductances in CFTR strongly suggest that CFTR may have two distinct pores. Thus, like ClC0, CFTR is likely to be a double-barreled ion channel, with the first half of CFTR forming the large conductance and the second half forming the small conductance. Quinton suggested in 1983 (1) that ClϪ transport is defective in cystic fibrosis and 6 years later, cloning of the cystic fibrosis gene (2) and subsequent studies showed that CFTR 1 is indeed a chloride channel (3-5). CFTR contains an anion selective pore with a linear I/V relationship (6, 7). In excised inside-out patches, wild-type CFTR exhibits two conductances, a main conductance of between 9 and 11 pS and a smaller conductance of approximately 4.0 pS. The selectivity of the main conductance is BrMany investigators have used mutagenesis to create CFTR cDNAs containing both naturally occurring and artificial mutations and truncated forms to CFTR to study the properties of the channel pore region. These studies as well as those using cysteine-scanning mutagenesis have pinpointed amino acid residues in the transmembrane segment 6 of TMD1 as critical to forming the main conductive pore of CFTR (3,4,8). We showed previously that TNR CFTR (comprising the first transmembrane domain, the first nucleotide binding domain, and the R domain) can form a functional chloride channel with characteristics approach...

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