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

Abstract

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel Na_V1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat Na_V1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3nM) and (Ca²⁺)₄-CaM (~85nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca²⁺)₄-CaM, NMR demonstrated that Na_V1.2 IQ motif peptide (Na_V1.2_IQp) exclusively made contacts with C-domain residues of CaM (CaM_C). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca²⁺)₂-CaM_C bound to Na_V1.2_IQp. The polarity of (Ca²⁺)₂-CaM_C relative to the IQ motif was opposite to that seen in apo CaM_C-Na_v1.2_IQp (2KXW), revealing that CaM_C recognizes nested, anti-parallel sites in Na_v1.2_IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaM_N allowing interactions with non-IQ Na_V1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.

Keywords: Allostery; Binding; Biosensor; FRET; Free energy; Linkage; Molecular recognition; NMR; Sodium channels; Thermodynamics; Titration.