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...
To test the role of nucleotide-binding fold (NBF) 2 and its interaction with the regulatory
We report here distinct rectification of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel reconstituted in lipid bilayer membranes. Under the symmetrical ionic condition of 200 mM KCl (with 1 mM MgCl2 in cis intracellular and 0 MgCl2 in trans extracellular solutions, pH in both solutions buffered at 7.4 with 10 mM HEPES), the inward currents (intracellular-->extracellular chloride movement) through a single CFTR channel were approximately 20% larger than the outward currents. This inward rectification of the CFTR channel was mediated by extracellular divalent cations, as the linear current-voltage relationship of the channel could be restored through the addition of millimolar concentrations of MgCl2 or CaCl2 to the trans solution. The dose responses for [Mg]zero and [Ca]zero had half-dissociation constants of 152 +/- 72 microM and 172 +/- 40 microM, respectively. Changing the pH buffer from HEPES to N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid did not alter rectification of the CFTR channel. The nonlinear conductance property of the CFTR channel seemed to be due to negative surface charges on the CFTR protein, because in pure neutral phospholipid bilayers, clear rectification of the channel was also observed when the extracellular solution did not contain divalent cations. The CFTR protein contains clusters of negatively charged amino acids on several extracellular loops joining the transmembrane segments, which could constitute the putative binding sites for Ca and Mg.
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