The Neuronal Voltage-Dependent Sodium Channel Type II IQ Motif Lowers the Calcium Affinity of the C-Domain of Calmodulin^†

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is Na_V1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the Na_V1.2 C-terminal tail (residues 1901−1938) [Mori, M., et al. (2000) Biochemistry 39, 1316−1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and Na_V1.2 at varying calcium concentrations. At 1 mM MgCl₂, both full-length CaM (CaM_1-148) and a C-domain fragment (CaM_76-148) exhibit tight (nanomolar) calcium-independent binding to the Na_V1.2 IQ motif, whereas an N-domain fragment of CaM (CaM_1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the Na_V1.2 IQ peptide showed that the peptide reduced the calcium affinity of the CaM C-domain sites (III and IV) without affecting the N-domain sites (I and II) significantly. This leads us to propose that the CaM C-domain mediates constitutive binding to the Na_V1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium. This contrasts with the CaM-binding domains of voltage-dependent Ca²⁺ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C-domain of CaM.