Xiao-Dong Zhang, Wei-Ping Yu, Tsung-Yu Chen
2010
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Journal
Biophysical Journal
Abstract
Previous experiments from our laboratory showed that the negatively-charged methanethiosulfonate (MTS) reagent, 2-sulfonatoethyl MTS (MTSES) modified a cysteine residue at the Y512 position of CLC-0 faster than the positively-charged 2-(trimethylammonium)ethyl MTS (MTSET). This observation suggested a hypothesis that the pore of CLC-0 may be built with a positive intrinsic pore potential. The hypothesis, however, is challenged by our most recent finding that the preference for the negatively charged MTS reagent is significantly reduced when the cysteine is placed at a deeper pore position, E166. In this study, we examine the discrepancy in the preference for charged MTS reagents between the Y512C and E166C mutants. We find that the inhibition of E166C by intracellularly-applied MTS reagents is contaminated by the modification of an endogenous cysteine because MTS modification rates of the E166A and E166C mutants are similar to each other. We identify that C229, which is located at the dimer interface of the channel, is the endogenous cysteine responsible for this contamination. After C229 is mutated, CLC-0 resumes a preference for selecting the negatively-charged over the positively-charged MTS reagents in modifying E166C, re-confirming the idea of a positive intrinsic potential in the pore. Our study also suggests a communication between the pore region near E166 and the dimer interface near C229 because the inhibition of the channel due to the modification of C229 is dependent upon the amino acid placed at position 166.