Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin:: Does VDSC acquire calmodulin-mediated Ca2+-sensitivity?

The voltage-dependent sodium channel (VDSC) interacts with intracellular molecules to modulate channel properties and localizations in neuronal cells. To study protein interactions, we applied yeast two-hybrid screening to the cytoplasmic C-terminal domain of the main pore-forming alpha-subunit. We found a novel interaction between the C-terminal domain and calmodulin (CaM). By two-hybrid interaction assays, we specified the interaction site of VDSC in a C-terminal region, which is composed of 38 amino acid residues and contains both IQ-Like and Baa motifs. Using a fusion protein of the C-terminal domain, we showed that interaction with CaM occurred in the presence and absence of Ca2+. Two synthetic peptides, each covering the IQ-like (NaIQ) or the Baa motifs (NaBaa), were used to examine the binding property by a gel mobility shift assay. Although the NdIQ and NaBaa sequences are overlapped, NaBaa binds only to Ca2+-bound Ca2+CaM, whereas NaIQ binds to both Ca2+CaM and Ca2+-free apoCaM. Fluorescence spectroscopy of dansylated CaM showed Ca2+-dependent spectral changes not only for NaBaa CaM but also for NaIQ . CaM. The results, taken together with other results, indicate that whereas the NaBaa CaM complex is formed in a Ca2+-dependent manner, the NaIQ . CgM complex has two conformational states, distinct with respect to the peptide binding site and the CaM conformation, depending on the Ca2+ concentration. These observations suggest the possibility that VDSC is functionally modulated through the direct CaM interaction and the Ca2+-dependent conformational transition of the complex.