Abstract
OBJECTIVE: To investigate the effect of calmodulin (CaM) and its mutants on binding to voltage-gated Na channel isoleucine-glutamine domain (Na(V)1.2 IQ). METHODS: The cDNA of Na(V)1.2 IQ was constructed by PCR technique, CaM mutants CaM(12), CaM(34) and CaM(1234) were constructed with Quickchange(TM) site-directed mutagenesis kit (QIAGEN). The binding of Na(V)1.2 IQ to CaM and CaM mutants under calcium and calcium free conditions were detected by pull-down assay. RESULTS: Na(V)1.2 IQ and CaM were bound to each other at different calcium concentrations, while GST alone did not bind to CaM. The binding affinity of CaM and Na(V)1.2 IQ at [Ca(2+)]-free was greater than that at 100 nmol/L [Ca(2+)] (P < 0.05). In the absence of calcium, the binding amount of CaM wild-type to Na(V)1.2 IQ was greater than that of its mutant, and the binding affinity of CaM(1234) to Na(V)1.2 IQ was the weakest among the three mutants (P < 0.05). CONCLUSIONS: The binding ability of CaM and CaM mutants to Na(V)1.2 IQ is Ca(2+)-dependent. This study has revealed a new mechanism of Na(V)1.2 regulated by CaM, which would be useful for the study of ion channel related diseases.