TY - JOUR
T1 - Variable reactivity of an engineered cysteine at position 338 in cystic fibrosis transmembrane conductance regulator reflects different chemical states of the thiol
AU - Liu, Xuehong
AU - Alexander, Christopher
AU - Serrano, Jose
AU - Borg, Erik
AU - Dawson, David C.
PY - 2006/3/24
Y1 - 2006/3/24
N2 - 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. One of the altered states displays reactivity toward thiols like dithiothreitol and 2-mercaptoethanol as well as mixed disulfides (methanethiosulfonate reagents: MTSET+, MTSES-) and an alkylating agent (iodoacetamide). The other altered state is unreactive. The phenotype associated with the reactive, altered state could be replicated by exposing oocytes expressing T338C CFTR to CuCl2, but not by glutathionylation or nitrosylation of the thiol or by oxidation with hydrogen peroxide. The results are consistent with the hypothesis that substituting a cysteine at 338 can create an adventitious metal binding site. Metal liganding alters thiol reactivity and may, in some cases, catalyze oxidation of the thiol to an unreactive form such as a sulfinic or sulfonic acid.
AB - 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. One of the altered states displays reactivity toward thiols like dithiothreitol and 2-mercaptoethanol as well as mixed disulfides (methanethiosulfonate reagents: MTSET+, MTSES-) and an alkylating agent (iodoacetamide). The other altered state is unreactive. The phenotype associated with the reactive, altered state could be replicated by exposing oocytes expressing T338C CFTR to CuCl2, but not by glutathionylation or nitrosylation of the thiol or by oxidation with hydrogen peroxide. The results are consistent with the hypothesis that substituting a cysteine at 338 can create an adventitious metal binding site. Metal liganding alters thiol reactivity and may, in some cases, catalyze oxidation of the thiol to an unreactive form such as a sulfinic or sulfonic acid.
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U2 - 10.1074/jbc.M512458200
DO - 10.1074/jbc.M512458200
M3 - Article
C2 - 16436375
AN - SCOPUS:33646363584
SN - 0021-9258
VL - 281
SP - 8275
EP - 8285
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 12
ER -