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Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
15:28

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

Published on: October 1, 2010

Inward rectification in ClC-0 chloride channels caused by mutations in several protein regions

U Ludewig1, T J Jentsch, M Pusch

  • 1Center for Molecular Neurobiology (ZMNH), Hamburg University, D-20246 Hamburg, Germany.

The Journal of General Physiology
|August 1, 1997
PubMed

Insights

The study reveals that specific mutations in ClC-0 and ClC-1 chloride channels alter their voltage-dependent gating mechanisms, suggesting similar gating principles across these channels. This challenges the notion of an intrinsic voltage sensor in ClC-channels.

Area of Science:

  • Molecular Biology
  • Biophysics
  • Ion Channel Physiology

Background:

  • ClC-type chloride channels exhibit voltage-dependent gating.
  • ClC-0 and ClC-1 channels show distinct gating mechanisms, with ClC-1 proposed to have an intrinsic voltage sensor.
  • A mutation in ClC-1 (D136G) informed its gating model.

Purpose of the Study:

  • To investigate the functional effects of mutating residue D70 in ClC-0, corresponding to D136 in ClC-1.
  • To explore the role of specific residues in the voltage-dependent gating of ClC-0 and ClC-1 channels.
  • To determine if an intrinsic voltage sensor is necessary for ClC-channel voltage dependence.

Main Methods:

  • Site-directed mutagenesis of ClC-0 at residue D70 (D70G, D70E) and other positions (K165R, H472K, S475T, E482D, T484S, T484Q).
  • Electrophysiological recordings to analyze channel gating properties.
  • Comparison of mutant phenotypes with wild-type ClC-0 and ClC-1 (D136G).

Main Results:

  • Mutations D70G and D70E in ClC-0 induced an inwardly rectifying phenotype, similar to ClC-1 (D136G).
  • Several other mutations at different positions in ClC-0 also resulted in a similar phenotype.
  • The T484S mutation's gating could be partially restored using external perchlorate (ClO4-).

Conclusions:

  • Gating mechanisms in ClC-0 and ClC-1 channels operate via similar principles.
  • The negative charge at position 70 in ClC-0 is not solely responsible for voltage sensitivity.
  • There is no requirement to postulate an intrinsic voltage sensor for voltage dependence in ClC-channels.

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