Journal
JOURNAL OF GENERAL PHYSIOLOGY
Volume 122, Issue 2, Pages 147-159Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.200308845
Keywords
MTS modification; SCAM; charge selection; state dependence
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Funding
- NIGMS NIH HHS [R01 GM065447, GM-65447, R56 GM065447] Funding Source: Medline
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ClC channels are a family of protein molecules containing two ion-permeation pores. Although these transmembrane proteins are important for a variety of physiological functions, their molecular operations are only superficially understood. High-resolution X-ray crystallography techniques have recently revealed the structures of two bacterial ClC channels, but whether vertebrate ClC channel pores are similar to those of bacterial homologues is not clear. To study the pore architecture of the Torpedo ClC-0 channel, we employed the substituted-cysteine-accessibility method (SCAM) and used charged methane thiosulfonate (MTS) compounds to modify the introduced cysteine. Several conclusions were derived from this approach. First, the MTS modification pattern from Y512C to E526C in ClC-0, which corresponds to residues forming helix R in bacterial ClC channels, is indeed consistent with the suggested helical structure. Second, the ClC-0 pore is more accessible to the negatively charged than to the positively charged NITS compound, a pore property that is regulated by the intrinsic electrostatic potential in the pore. Finally, attempts to modify the introduced cysteine at positions intracellular to the selectivity filter did not result in larger MTS modification rates for the open-state channel, suggesting that the fast gate of ClC-0 cannot be located at a position intracellular to the Cl- selectivity filter. Thus, the proposal that the glutamate side chain is the fast gate of the channel is applicable to ClC-0, revealing a structural and functional conservation of ClC channels between bacterial and vertebrate species.
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