Cysteine modification of a putative pore residue in ClC-0: implication for the pore stoichiometry of ClC chloride channels.
The ClC channel family consists of chloride channels important for various physiological functions. Two members in this family, ClC-0 and ClC-1, share approximately 50-60% amino acid identity and show similar gating behaviors. Although they both contain two subunits, the number of pores present in the homodimeric channel is controversial. The double-barrel model proposed for ClC-0 was recently challenged by a one-pore model partly based on experiments with ClC-1 exploiting cysteine mutagenesis followed by modification with methanethiosulfonate (MTS) reagents. To investigate the pore stoichiometry of ClC-0 more rigorously, we applied a similar strategy of MTS modification in an inactivation-suppressed mutant (C212S) of ClC-0. Mutation of lysine 165 to cysteine (K165C) rendered the channel nonfunctional, but modification of the introduced cysteine by 2-aminoethyl MTS (MTSEA) recovered functional channels with altered properties of gating-permeation coupling. The fast gate of the MTSEA-modified K165C homodimer responded to external Cl(-) less effectively, so the P(o)-V curve was shifted to a more depolarized potential by approximately 45 mV. The K165C-K165 heterodimer showed double-barrel-like channel activity after MTSEA modification, with the fast-gating behaviors mimicking a combination of those of the mutant and the wild-type pore, as expected for the two-pore model. Without MTSEA modification, the heterodimer showed only one pore, and was easier to inactivate than the two-pore channel. These results showed that K165 is important for both the fast and slow gating of ClC-0. Therefore, the effects of MTS reagents on channel gating need to be carefully considered when interpreting the apparent modification rate.
Lin
C W
CW
Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.
Chen
T Y
TY
D000818:Animals; D018118:Chloride Channels / Q000187:drug effects* / Q000502:physiology; D002712:Chlorides / Q000494:pharmacology; D003545:Cysteine / Q000187:drug effects / Q000235:genetics*; D019281:Dimerization; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology*; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D014981:Xenopus
C120501:CLC-0 chloride channel protein, Torpedo californica; C086321:CLC-1 channel; D018118:Chloride Channels; D002712:Chlorides; D007202:Indicators and Reagents; C093171:methanethiosulfonate ethylammonium; D005020:Ethyl Methanesulfonate; D003545:Cysteine