Slow gating of gap junction channels and calmodulin

Area of Science:

• Cellular Biology
• Biophysics
• Molecular Biology

Background:

• Connexin32 (Cx32) gap junctions form channels regulating cell-to-cell communication.
• Previous studies indicated unusual voltage sensitivity in Cx32 COOH-terminus mutants.
• Calmodulin (CaM) is implicated in gap junction channel function.

Purpose of the Study:

• To investigate the gating properties of new Cx32 mutants in cytoplasmic-loop and COOH-terminus regions.
• To test the hypothesis that a CaM-like molecule acts as a pore plug (cork gating model).
• To determine the role of CaM in Cx32 channel voltage and CO2 sensitivity.

Main Methods:

• Creation and functional analysis of four new Cx32 mutants (ML/NN+3R/N, 3R/N, ML/NN, ML/EE).
• Heterotypic channel formation in oocytes between Cx32 wild type and mutants.
• Measurement of junctional conductance (G(j)) under varying transjunctional voltage (V(j)).
• Assessment of CO2-induced uncoupling and CaM expression inhibition.

Main Results:

• Mutant Cx32 channels displayed slow, voltage-dependent gating, with conductance increasing at positive V(j) and decreasing at negative V(j).
• Positive V(j) at the mutant side reversed CO2-induced uncoupling.
• Inhibition of CaM expression significantly reduced V(j) and CO2 sensitivities in two key mutants.

Conclusions:

• The observed slow gating in Cx32 mutants supports a CaM-mediated 'cork gating' mechanism.
• Calmodulin plays a crucial role in both the slow voltage-dependent and chemical gating of Cx32 channels.