Intracellular Cysteines of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulate Channel Gating

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette superfamily, is a cAMP-activated chloride channel. CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. We found that whole-cell CFTR-dependent Cl- currents in Xenopus laevis oocytes were sensitive to HgCl₂, suggesting that modification of endogenous cysteines alters channel activity. To understand better this phenomenon, site-directed mutagenesis was employed to generate both individual cysteine replacements and a version of the molecule with no cysteines in the hydrophobic sector. Each mutant displayed a forskolin/IBMX-activated Cl^- conductance similar to wild type, indicating that none of the cysteines located within the TMDs is essential. Subsequent single-channel analysis of inside-out patches excised from HEK293 cells expressing either cysteine-less or wild-type CFTR showed that intracellular application of a membrane impermeant sulphydryl reagent, p-chloromercuribenzosulfonate (PCMBS), significantly reduced open probability without affecting ion selectivity or conductance. The cysteine-less molecule also acquired a voltage-dependent sensitivity to extracellular PCMBS not observed in the wild type, perhaps due to a more flexible conformation that allowed PCMBS access to the intracellular surface. Together, these experiments suggest that endogenous intracellular cysteines, located primarily within the NBDs and/or R domain, influence channel gating.

Mercury and zinc differentially inhibit shark and human CFTR orthologues: involvement of shark cysteine 102

“…In other membrane protein systems, pCMBS sensitivity has correlated with the presence of exofacial cysteine residues (31,65). Cysteine residues in the R-domain, sensitive to 10-to 100-fold higher concentrations of mercury, were recently suggested as sites influencing channel gating in human CFTR Cl Ϫ channels (33). In agreement with our findings was the lack of significant inhibition of hCFTR by pCMBS (33).…”

“…Cysteine residues in the R-domain, sensitive to 10-to 100-fold higher concentrations of mercury, were recently suggested as sites influencing channel gating in human CFTR Cl Ϫ channels (33). In agreement with our findings was the lack of significant inhibition of hCFTR by pCMBS (33). The striking differences in sensitivity to HgCl 2 , zinc, and pCMBS in oocytes expressing sCFTR and hCFTR, and the presence of unique cysteines not found in the human protein, reinforce the idea that cysteine thiolates are the site(s) of action of mercury as well as pCMBS and zinc.…”

Insight in eukaryotic ABC transporter function by mutation analysis

“…Using synthetic peptides corresponding to TM4, this residue together with D232 and Q237 have been shown to mediate helix–helix interactions [173]. Other endogenous cysteine residues located within the NBDs and/or R domain govern channel gating by specifically modulating open probability [174]. Mutating G314 and V317 (TM5) and S1118 (TM11) affected both anion permeation and channel gating, suggesting that they also contribute to the pore [50,175,176].…”

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

“…c Channel blockers were prepared as noted [11] and added to both sides of the membrane; final concentrations were 1 mM glibenclamide, 1 mM DPC, 5 mM DIDS, and 10 mM glutathione.…”

“…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].…”