Human voltage-gated sodium channel NaV1.2 has a single pore-forming α-subunit and two transmembrane β-subunits. Expressed primarily in the brain, NaV1.2 is critical for initiation and propagation of action potentials. Milliseconds after the pore opens, sodium influx is terminated by inactivation processes mediated by regulatory proteins including calmodulin (CaM). Both calcium-free (apo) CaM and calcium-saturated CaM bind tightly to an IQ motif in the C-terminal tail of the α-subunit. Our thermodynamic studies and solution structure (2KXW) of a C-domain fragment of apo 13C,15N- CaM (CaMC) bound to an unlabeled peptide with the sequence of rat NaV1.2 IQ motif showed that apo CaMC (a) was necessary and sufficient for binding, and (b) bound more favorably than calcium-saturated CaMC. However, we could not monitor the NaV1.2 residues directly, and no structure of full-length CaM (including the N-domain of CaM (CaMN)) was determined. To distinguish contributions of CaMN and CaMC, we used solution NMR spectroscopy to assign the backbone resonances of a complex containing a 13C,15N-labeled peptide with the sequence of human NaV1.2 IQ motif (NaV1.2IQp) bound to apo 13C,15N-CaM or apo 13C,15N-CaMC. Comparing the assignments of apo CaM in complex with NaV1.2IQp to those of free apo CaM showed that residues within CaMC were significantly perturbed, while residues within CaMN were essentially unchanged. The chemical shifts of residues in NaV1.2IQp and in the C-domain of CaM were nearly identical regardless of whether CaMN was covalently linked to CaMC. This suggests that CaMN does not influence apo CaM binding to NaV1.2IQp.
Keywords: Allostery; Domain interactions; EF-hand protein; Molecular recognition; Voltage-gated sodium channel.