Variable Reactivity of an Engineered Cysteine at Position 338 in Cystic Fibrosis Transmembrane Conductance Regulator Reflects Different Chemical States of the Thiol

Abstract

In a previous study of T338C CFTR (cystic fibrosis transmembrane conductance regulator) we found that protons and thiol-directed reagents modified channel properties in a manner consistent with the hypothesis that this residue lies within the conduction path, but the observed reactivity was not consistent with the presence of a single thiolate species in the pore. Here we report results consistent with the notion that the thiol moiety can exist in at least three chemical states, the simple thiol, and two altered states. The technique of cysteine scanning mutagenesis has been widely used to probe the structure and functional properties of ion channels (1-8), including the cystic fibrosis transmembrane conductance regulator (CFTR) 4 (9 -11). The strategy is based on the notion that the reactive thiolate anion can interact with a variety of reagents including mixed disulfides (8,(12)(13)(14), alkylating reagents (12, 15, 16), and metals (17-20). In such experiments a typical first step is to screen various cysteine-substituted constructs for reactivity using, for example, reagents such as MTSET ϩ or MTSES Ϫ that react with the thiolate anion by means of thiol-disulfide exchange (14). The result is a pattern of reactivity, usually portrayed as the relative increase, decrease, or no change in conductance. In such an assay, if exposure to a reagent results in no change in conductance, it might be thought that the cysteine in question was not accessible to the reagents. It is recognized by practitioners, however, that a failure to respond could also reflect either the lack of any functional consequence of the reaction or altered reactivity of the engineered cysteine in its non-natural, protein environment. Although the latter possibility is generally recognized, potential mechanisms have not been studied in any detail (7,8,21). We report here evidence that a cysteine substituted into transmembrane segment 6 of CFTR can undergo spontaneous changes in its reactivity toward thiol-directed reagents that can produce conflicting results in a screening assay. These effects appear to be attributable to changes in the chemical state of the cysteine thiol. The goal of the experiments described here was to use thiol reactivity to operationally define different states of the cysteine at position 338 and to compare altered reactivity with that induced experimentally. The results are consistent with the hypothesis that the engineered cysteine is a component of an adventitious metal binding site that exhibits a high affinity for copper. MATERIALS AND METHODSMutagenesis and in Vitro Transcription-The methods used for mutagenesis and in vitro transcription were similar to those reported previously (11,22). Briefly, CFTR mutants were generated using the QuikChange TM site-directed mutagenesis kit from Stratagene. The sequences in the region of the mutation and in the entire PCR-generated region were confirmed by direct DNA sequencing.The T338C CFTR mutants used in this study were generated on two different CFTR backgrounds, either the ...

Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking

“…These results indicate that post-translational modification of the Cys-substituted residue in either Q45C-DSL or L290C-DSL is not due to the formation of a disulfide bond with one of the native Cys residues. Rather, the data are consistent with modification by endogenous thiols similar to what occurred when a Cys residue was substituted for Thr 338 of the cystic fibrosis transmembrane conductance regulator (36). Most important, the modifications of Q45C and L290C mutants appeared to be reversible with DTT reduction.…”

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Salt Bridge Switching from Arg290/Glu167 to Arg290/ATP Promotes the Closed-to-Open Transition of the P2X2 Receptor

“…These data suggest that the two cysteines can spontaneously form a disulfide bond. Similarly, variable levels of spontaneous disulfide bond formation have been previously observed in different batches of oocytes (Liu et al, 2006). Neither H 2 O 2 nor DTT produced a statistically significant change in the current amplitudes (Fig.…”

A conserved salt bridge critical for GABA A receptor function and loop C dynamics

“…For a subset of oocytes expressing ␤E153C-␤K196C, application of DTT to naïve oocytes significantly increased EC 50 GABA current amplitudes (data not shown) suggesting that under certain conditions the two cysteines are spontaneously crosslinked. The variability in observing spontaneous disulfide bond formation between ␤E153C and ␤K196C is likely because of differences in the redox environment of different batches of oocytes (23). Disulfide bond formation induced by H 2 O 2 between ␤E153C and ␤K196C reduced GABA gated current and likely traps loop C in a position not favorable for receptor activation.…”