A novel binding site between the voltage-dependent calcium channel CaV1.2 subunit and CaVβ2 subunit discovered using a new analysis method for protein-protein interactions

We developed a new method to analyze protein-protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein-protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisomerase II toxin. Protein-protein interactions would affect toxin activity, resulting in colony formation. Using this novel system, we found a new binding site in the voltage-dependent calcium channel alpha 1 subunit (Ca(V)1.2) for the voltage-dependent calcium channel beta 2 subunit. Prokaryotic expression screening of the beta 2 subunit using an epitope library of Ca(V)1.2 resulted in two overlapping clones of the C-terminal sequence of Ca(V)1.2. In vitro overlay and immunoprecipitation analyses revealed preferential binding of the C-terminal sequences of Ca(V)1.2 and beta 2.

Automated summary

A new method was developed to analyze protein-protein interactions using a dual-inducible prokaryotic expression system. This method involved constructing a chimeric fusion toxin gene to evaluate protein-protein binding. The study identified a new binding site in a voltage-dependent calcium channel.