title: Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin: does VDSC acquire calmodulin-mediated Ca2+-sensitivity?
abstract: 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 Ca(2+). 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 NaIQ and NaBaa sequences are overlapped, NaBaa binds only to Ca(2+)-bound Ca(2+)CaM, whereas NaIQ binds to both Ca(2+)CaM and Ca(2+)-free apoCaM. Fluorescence spectroscopy of dansylated CaM showed Ca(2+)-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 Ca(2+)-dependent manner, the NaIQ.CaM complex has two conformational states, distinct with respect to the peptide binding site and the CaM conformation, depending on the Ca(2+) concentration. These observations suggest the possibility that VDSC is functionally modulated through the direct CaM interaction and the Ca(2+)-dependent conformational transition of the complex.
authors.affiliation: Department of Physiological Sciences, The Graduate University for Advanced Studies, National Institue for Physiological Sciences, Okazaki, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Mori
authors.forename: T
authors.initials: T
authors.lastname: Konno
authors.forename: T
authors.initials: T
authors.lastname: Ozawa
authors.forename: M
authors.initials: M
authors.lastname: Murata
authors.forename: K
authors.initials: K
authors.lastname: Imoto
authors.forename: K
authors.initials: K
authors.lastname: Nagayama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry / Q000235:genetics; D002147:Calmodulin / Q000378:metabolism* / Q000502:physiology; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D002147:Calmodulin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels