Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel NaV1.2

• NaV1.2 IQ motif binds apo and (Ca2 +)4-CaM tightly: how can Ca2 + binding trigger change?
• Direct measure of equilibrium Kd values: apo CaM (~ 3 nM) vs. (Ca2 +)4-CaM (~ 85 nM)
• Solution structure of (Ca2 +)2-CaMC bound to NaV1.2 IQ has “open” EF-hand conformation.
• 2KXW and 2M5E are first NMR structures of apo and Ca2 +-CaMC bound to same channel.
• CaM binds nested, anti-parallel sites in NaV1.2 IQ; Ca2 + binding to CaMC reverses its orientation.

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 (NaV1.2IQp) 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.

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