Locating the Anion-selectivity Filter of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channel

Abstract

The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel; the site and mechanism of charge selectivity is unknown. We previously reported that cysteines substituted, one at a time, for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353, in and flanking the sixth membrane-spanning segment (M6), reacted with charged, sulfhydryl-specific, methanethiosulfonate (MTS) reagents. We inferred that these residues are on the water-accessible surface of the protein and may line the ion channel. We have now measured the voltage-dependence of the reaction rates of the MTS reagents with the accessible, engineered cysteines. By comparing the reaction rates of negatively and positively charged MTS reagents with these cysteines, we measured the extent of anion selectivity from the extracellular end of the channel to eight of the accessible residues. We show that the major site determining anion vs. cation selectivity is near the cytoplasmic end of the channel; it favors anions by ∼25-fold and may involve the residues Arg347 and Arg352. From the voltage dependence of the reaction rates, we calculated the electrical distance to the accessible residues. For the residues from Leu333 to Ser341 the electrical distance is not significantly different than zero; it is significantly different than zero for the residues Thr351 to Gln353. The maximum electrical distance measured was 0.6 suggesting that the channel extends more cytoplasmically and may include residues flanking the cytoplasmic end of the M6 segment. Furthermore, the electrical distance calculations indicate that R352C is closer to the extracellular end of the channel than either of the adjacent residues. We speculate that the cytoplasmic end of the M6 segment may loop back into the channel narrowing the lumen and thereby forming both the major resistance to current flow and the anion-selectivity filter.

Identification of Residues Lining the Translocation Pore of Human AE1, Plasma Membrane Anion Exchange Protein

“…Failure to observe complete transport inhibition with both MTSEA and pCMBS reflects (i) that the assay measures only covalent inhibition, (ii) chemical reaction only occurred for 8 min, and (iii) only moderate concentrations of sulfhydryl reagents were used to prevent nonspecific effects on the cell. In studies to identify pore-lining residues of channels, a maximum inhibition of 25-40% was also observed (25,26). Introduced cysteine mutants were defined as those inhibited by sulfhydryl reagents if residual activity after inhibition was less than 100%.…”

“…Derivatization of a cysteine with a methanethiosulfonate will impair ion conductance through steric blockage of a con-fined ion translocation pore. Pore-lining residues in the cystic fibrosis chloride channel, CFTR (23)(24)(25), ␥-aminobutyric acid receptor chloride channel (26) and acetylcholine receptor sodium channel (27,28) have been identified in this way. Differential sensitivity to inhibition by methanethiosulfonate compounds has also identified residues of the transport pathway in the glutamate transport protein GLT-1 (29), sodium glucose cotransporter (30), and sodium/calcium exchanger (31).…”

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

“…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.…”

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

“…In CFTR, unlike other ABC transporters, a third domain, termed the regulatory (R) domain, is located between the two half molecules. Current evidence suggests that the TMDs define the CFTR chloride channel, while the NBDs and the R domain mediate channel gating [3][4][5][6][7][8][9][10][11] Although CFTR is glycosylated, there is currently no evidence indicating that the presence of carbohydrate affects CFTR structure or function [12]. Consistent with this presumption, expression of human CFTR in Sf9 insect cells results in appearance of the 140 kD core polypeptide -containing little or no glycosylation -that mediates a newly acquired anion permeability with the electrophysiological signature of CFTR [13] [14,15].…”