A novel binding site between the voltage-dependent calcium channel Ca(V)1.2 subunit and Ca(V)β2 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 α1 subunit (Ca(V)1.2) for the voltage-dependent calcium channel β2 subunit. Prokaryotic expression screening of the β2 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 β2.