Multi-ion pore behaviour in the CFTR chloride channel

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)-and ATP-regulated chloride channel, whose gating process involves intra-or intermolecular interactions among the cytosolic domains of the CFTR protein.Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP-and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions. CFTR1 is a multi-functional protein, which provides the pore of a linear conductance chloride channel (1-5) and also functions to regulate other membrane proteins (6, 7). Mutations in CFTR leading to defective regulation or transport of chloride ions across the apical surface of epithelial cells are the primary cause of the genetic disease of cystic fibrosis (8 -10). Comprehensive genotype-phenotype studies have indicated possible contribution of protein-protein interactions to the severity of the disease (11), but little is known on the stoichiometry of CFTR as a chloride channel.The native CFTR chloride channel is activated by PKAphosphorylation of serine residues in its regulatory or R domain and then gated by binding and hydrolysis of ATP by the nucleotide binding folds (12). The actual pore of the chloride channel is presumably formed by portions of the two membrane spanning domains of CFTR, each consisting of six transmembrane segments (13), with an ohmic conductance of ϳ8 pS in 200 mM KCl solution (14,15). An early study by Rich et al. (16) showed that deletion of amino acids 708 -835 from the R domain (⌬R) renders the CFTR channel PKA independent. The open probability of ⌬R-CFTR is approximately one-third that of the wt channel and does not increase upon PKA phosphorylation (17,18). Based on these different gating properties of the wt and ⌬R channels, we set out to test the intermolecular interactions of CFTR by constructing tandem cDNAs between the wt-CFTR and the ⌬R-CFTR. Our rationale is as follows. If a monomer of CFTR is sufficient to form an 8-pS chloride channel, the dimeric CFTR molecules would form either a 16-pS chloride channel or two 8-pS chloride channels that may gate independently or together. On the other hand, if two CFTR molecules are required to function as a chloride channel, we expected the tandem construct to form a single 8-pS chloride channel, provided that the linker sequence does not affect the CFTR channel function. Furthermore, we predicted that the wt-⌬R (or ⌬R-wt) channel should exhibit mixed properties of the wt and ⌬R channels. EXPERIMENTAL PROCEDURESSubcloning of CFTR cDNAs-The wild type and ⌬R (708 -835) CFTR cDNAs were cloned into the NheI/XhoI sites of the pCEP4 expression vec...

Section: A-s-l-v-p-r-g-s-g-g-g-g) As Shown Inmentioning

confidence: 87%

Section: Cftr As a Dimer 7628mentioning

confidence: 99%

Section: Cftrmentioning

confidence: 99%

See 1 more Smart Citation

A Single Conductance Pore for Chloride Ions Formed by Two Cystic Fibrosis Transmembrane Conductance Regulator Molecules

Zerhusen

Zhao

Xie

et al. 1999

“…Patch-clamp Studies of CL3 Mutants-Single CFTR channels were recorded from inside-out patches excised from stably expressing CHO cells, according to Tabcharani et al (1991) and Tabcharani et al (1993). Following patch excision, channels were activated by exposing the cytoplasmic face of the patch to 75 nM catalytic subunit of PKA (Promega) plus 1 mM Na 2 ATP.…”

Section: Methodsmentioning

confidence: 99%

Cytoplasmic Loop Three of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Regulation of Chloride Channel Activity

Seibert

Linsdell

Loo

et al. 1996

Self Cite

104

To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three pointmutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Chinese hamster ovary cell lines stably expressing wildtype CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein. Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26°C) or incubated in the presence of 10% glycerol. For all mutants, the open probability (P 0 ) of the CFTR channels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the P 0 , whereas the H949Y mutation doubled the P 0 relative to wild-type. The changes in P 0 predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range ؎80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore.

“…Each of these residues was shown to be important in the Cl Ϫ conductance of the 9-pS pore of CFTR (16,17). In a previous report (18), we tested the hypothesis that insertion of arginine point mutations together could severely affect the Cl Ϫ conductance within CFTR.…”

Section: Single-channel Current Characteristics Of Cftr-mentioning

confidence: 99%

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

Yue

Devidas

Guggino

2000

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...