Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel NaV1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat NaV1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3 nM) and (Ca2+)4-CaM (~85 nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca2+)4-CaM, NMR demonstrated that NaV1.2 IQ motif peptide (NaV1.2|Qp) exclusively made contacts with C-domain residues of CaM (CaMC). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca2+)2-CaMC bound to NaV1.2IQp. The polarity of (Ca2+)2-CaMC relative to the IQ motif was opposite to that seen in apo CaMC-Nav1.2IQp (2KXW), revealing that CaMC recognizes nested, anti-parallel sites in Nav1.2IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaMN allowing interactions with non-IQ NaV1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.

Na V 1.2 EFL domain allosterically enhances Ca 2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD Graphical abstract Highlights d In solution, only the IQ motif of Na V 1.2 CTD(1,777-1,937) contacts CaM ± Ca 2+ d Ca 2+ changes interfaces of CaM + Na V 1.2-IQ p and CaM + Na V 1.2-CTD complexes identically d Ca 2+ binds to N-domain sites of CaM + Na V 1.2-CTD preferentially relative to free CaM d Apo pathogenic CaM mutants bind the Na V 1.2 CTD as well or better than WT CaM

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Voltage-gated sodium channels (NaV) found in excitable cells are responsible for the rising phase of action potentials. These multi-domain transmembrane proteins are regulated by calmodulin (CaM), a highly conserved eukaryotic protein that mediates many calcium-triggered signaling events. Inactivation of sodium channels depends on CaM-mediated feedback during repolarization. In the neuronal sodium channel NaV1.2, CaM binds at least two well-separated sites: an intracellular ''inactivation'' loop between domains DIII and DIV, and an IQ motif [IQRAYRRYLLK] in the cytosolic C-terminal tail. The IQ motif is hypothesized to recruit calcium-free (apo) CaM, making it available to move to the III-IV linker after an influx of calcium. Despite a high degree of sequence identity, the equilibrium constants for CaM binding to nine human NaV IQ motifs span more than 3 orders of magnitude. Apo CaM binds to the NaV1.2 IQ motif with a dissociation constant (Kd) of~6 nM, while the Kd for binding the NaV1.9 IQ motif is~4 mM. Mutational analysis within the IQ motif has not been sufficient to explain the full range of CaM-binding affinities observed for human NaV sequences. Thus, we hypothesized that isoform-specific differences in upstream sequences were making energetically significant contributions to the free energy of binding CaM to NaV1.2. The roles of these residues are being investigated by monitoring CaM binding to biosensors containing mutant sequences of sodium channels bracketed by auto-fluorescent proteins YFP and CFP. Residue-specific information obtained by NMR will provide structural insight into the contributions of residues in the binding interface formed by NaV IQ motif sequences binding to calmodulin from multiple eukaryotes. NIH R01 GM57001.

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