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

Abstract

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...

Imaging CFTR in its native environment

“…Images support functional studies in which tail-to-tail dimerization is associated with highest chloride channel conductance. Tandem linkage of two CFTR molecules (i.e., head to tail dimerization) as proposed in other functional studies [108,113] We expected to find two gold particles at each CFTR ring but, surprisingly, found only some of the ring-like structures connected to two gold particles. We do not know whether it is technically more difficult to detect CFTR rings when two (instead of one) gold particles are in close vicinity or whether the second antibody gold-labeled complex has been sterically obstructed.…”

“…Considering the molecular mass of 180 kDa for a CFTR monomer, the peak at 275 and 750 kDa could be a multimeric CFTR or CFTR forming clusters with other proteins. Dimerization of CFTR, as derived from electrical experiments, has been reported recently [113]. Mature CFTR has a short residence time at the cell surface; it is endocytosed within several minutes, but then, most is recycled back into the plasma membrane [52,72].…”

“…Freeze-fracture electromicroscopy of membranes of Xenopus oocytes in which CFTR was expressed revealed transmembrane domains with a crosssectional area that corresponds to a CFTR dimer [21]. From observations that expression of wild-type and mutant CFTR sequences linked in tandem resulted in channels with intermediate gating properties, Zerhausen et al [113] also concluded that two CFTR polypeptides form a functional CFTR. Additionally, linking at least two CFTR molecules via cytoplasmic C-terminal binding by CAP 70 potentiates CFTR chloride channel action [108].…”

Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging

“…Patch clamp analysis of constructs containing linked wild-type (WT) CFTRs or WT and mutant CFTRs suggested that two CFTR polypeptides form a single chloride conductance pathway (8). Conflicting data from reconstituted membranes containing WT and mutant CFTRs did not reveal intermediary conductance states, consistent with independently functioning CFTR monomers (9).…”

E3KARP Mediates the Association of Ezrin and Protein Kinase A with the Cystic Fibrosis Transmembrane Conductance Regulator in Airway Cells

“…the cross-sectional packing area corresponded to 24 transmembrane helices). In addition, when studied in planar lipid bilayers, the single channel properties derived from expression in HEK293 cells of a tandem repeat of two CFTRs were similar to the single channel properties obtained from expression of CFTR monomers (40). Co-expression of CFTR with deletion mutants of either E3KARP or EBP50/NHERF could be used to test further the hypothesis that these PDZ domain proteins can mediate the formation of CFTR homodimers.…”