Cysteine modification of a putative pore residue in ClC-0: Implication for the pore stoichiometry of ClC chloride channels

Chia Wei Lin, Tsung-Yu Chen

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Abstract

The ClC channel family consists of chloride channels important for various physiological functions. Two members in this family, ClC-0 and ClC-1, share ~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 ~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.

Original languageEnglish (US)
Pages (from-to)535-546
Number of pages12
JournalJournal of General Physiology
Volume116
Issue number4
DOIs
StatePublished - 2000
Externally publishedYes

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Keywords

  • 2-aminoethyl methanethiosulfonate
  • Chloride channel
  • ClC-0
  • Double barrel

ASJC Scopus subject areas

  • Physiology

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