Cysteine-independent inhibition of the CFTR chloride channel by the cysteine-reactive reagent sodium (2-sulphonatoethyl) methanethiosulphonate.
OBJECTIVE
Methanethiosulphonate (MTS) reagents are used extensively to modify covalently cysteine side chains in ion channel structure-function studies. We have investigated the interaction between a widely used negatively charged MTS reagent, (2-sulphonatoethyl) methanethiosulphonate (MTSES), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel.


METHODS
Patch clamp recordings were used to study a 'cys-less' variant of human CFTR, in which all 18 endogenous cysteine residues have been removed by mutagenesis, expressed in mammalian cell lines. Use of excised inside-out membrane patches allowed MTS reagents to be applied to the cytoplasmic face of active channels.


RESULTS
Intracellular application of MTSES, but not the positively charged MTSET, inhibited the function of cys-less CFTR. Inhibition was voltage dependent, with a K(d) of 1.97 mmol x L(-1) at -80 mV increasing to 36 mmol x L(-1) at +80 mV. Inhibition was completely reversed on washout of MTSES, inconsistent with covalent modification of the channel protein. At the single channel level, MTSES caused a concentration-dependent reduction in unitary current amplitude. This inhibition was strengthened when extracellular Cl(-) concentration was decreased.


CONCLUSIONS
Our results indicate that MTSES inhibits the function of CFTR in a manner that is independent of its ability to modify cysteine residues covalently. Instead, we suggest that MTSES functions as an open channel blocker that enters the CFTR channel pore from its cytoplasmic end to physically occlude Cl(-) permeation. Given the very widespread use of MTS reagents in functional studies, our findings offer a broadly applicable caveat to the interpretation of results obtained from such studies.
Li
M-S
MS
Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada.
Demsey
A F A
AF
Qi
J
J
Linsdell
P
P
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000502:physiology*; D006801:Humans; D008698:Mesylates / Q000494:pharmacology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D008698:Mesylates; D013439:Sulfhydryl Reagents; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C109153:(2-sulfonatoethyl)methanethiosulfonate; D003545:Cysteine