Redox Reagents and Divalent Cations Alter the Kinetics of Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

Abstract

Gating of the cystic fibrosis Cl؊ channel requires hydrolysis of ATP by its nucleotide binding folds, but how this process controls the kinetics of channel gating is poorly understood. In the present work we show that the kinetics of channel gating and presumably the rate of ATP hydrolysis depends on the species of divalent cation present and the oxidation state of the protein.With Ca 2؉ as the dominant divalent cation instead of Mg 2؉ , the open burst duration of the channel is increased approximately 20-fold, and this change is reversible upon washout of Ca 2؉ . In contrast, "soft" divalent cations such as Cd 2؉ interact covalently with cystic fibrosis transmembrane conductance regulator (CFTR). These metals decrease both opening and closing rates of the channel, and the effects are not reversed by washout. Oxidation of CFTR channels with a variety of oxidants resulted in a similar slowing of channel gating. In contrast, reducing agents had the opposite effect, increasing both opening and closing rates of the channel. In cell-attached patches, CFTR channels exhibit both oxidized and reduced types of gating, raising the possibility that regulation of the redox state of the channel may be a physiological mode of control of CFTR channel activity.The cystic fibrosis transmembrane conductance regulator (CFTR) 1 belongs to the superfamily of ATP-binding cassette transporters, members of which are characterized structurally by two ATP-binding motifs and functionally by their ability to couple ATP hydrolysis to transport of a substrate molecule across a membrane (1, 2). Although a role for CFTR in solute transport has been proposed (3-6), no substrate has yet been identified. CFTR remains unique among ATP-binding cassette transporters in that it couples ATP hydrolysis to the opening of a Cl Ϫ channel (6, 7), reviewed in (8). The role of ATP hydrolysis in the CFTR gating process was inferred initially from studies of CFTR gating in electrophysiological experiments using poorly hydrolyzable ATP analogs and transition state phosphate analogs (9 -12). Direct measurement of ATPase activity in purified reconstituted CFTR has confirmed that it functions as an ATPase, and the measured turnover rate of the phosphorylated form (0.5-1 s Ϫ1 ) is consistent with a model in which opening and closing of the CFTR channel gate is coupled directly to ATP hydrolysis (13).The role of ATP hydrolysis by the two nucleotide-binding folds (NBF1 and NBF2) in mediating channel gating has been elucidated by studies examining CFTR channels individually mutated in each of the NBFs (14 -16). These papers presented models in which two ATP hydrolytic events, one occurring at NBF1 and one at NBF2, are involved first in opening and then closing the CFTR channel. In one model a single cycle of ATP hydrolysis occurs at NBF1 to directly open the channel (8,14,16,17). Alternatively, a second model proposes that ATP hydrolysis at NBF1 converts the channel into a gatable state from which nucleotide binding at NBF2 can then open the channel (15). In both mod...

Cysteine Substitutions Reveal Dual Functions of the Amino-terminal Tail in Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

“…4) or the wild type channel. (Gating of wild type CFTR can be affected by DTT at millimolar concentrations of this reducing agent (19).) Thus, the simplest explanation of our data is that the MTS reagents affected CFTR gating by forming mixed disulfides with the engineered cysteines at positions 54 and 58 in the CFTR amino-terminal tail.…”

“…In addition, since cysteines can be modified with methanethiosulfonate (MTS) derivatives with different chemistries (16), such experiments have the potential to provide initial insights into the structural requirements for the involvement of the N-tail in channel gating. We were encouraged to attempt this approach because of reports that the CFTR channel is relatively insensitive to low concentrations of thiol-specific reagents (17)(18)(19). Our results indicate that modification of these cysteines with either uncharged or charged MTS reagents markedly and reversibly reduces channel opening rate in excised membrane patches.…”

Aqueous Accessibility to the Transmembrane Regions of Subunit c of the Escherichia coli F1F0 ATP Synthase

“…In the experiment shown in A, unmodified subunit c accounts for 12% of the total subunit c in the experiment shown. channels and in the cystic fibrosis transmembrane conductance regulator (35,36). Cd 2ϩ has an ionic radius similar to that of Ag ϩ (0.95 and 1.15 Å, respectively), and Cd 2ϩ , like Ag ϩ , is a soft Lewis acid that forms a covalent bond with the thiolate form of cysteine (37).…”

Vitamin C controls the cystic fibrosis transmembrane conductance regulator chloride channel

“…Second, vitamin C is well known for its properties as an antioxidant, and we considered the notion that ascorbate activated CFTR directly by modifying its redox state. Previous work has shown that redox reagents alter the kinetics of CFTR gating such that reducing conditions (e.g., 2-mercaptoethanol and dithiotreitol) speed up gating and increase the open probability of CFTR (45), whereas oxidizing conditions slow gating of CFTR, presumably through cysteine residues present in the nucleotide-binding domains of CFTR (46). A variety of reducing and sulfhydryl-modifying agents activate CFTR in a similar manner.…”