Backbone resonance assignments of complexes of human voltage-dependent sodium channel Na1.2 IQ motif peptide bound to apo calmodulin and to the C-domain fragment of apo calmodulin.

Human voltage-gated sodium channel Na1.2 has a single pore-forming α-subunit and two transmembrane β-subunits. Expressed primarily in the brain, Na1.2 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 C,N- CaM (CaM) bound to an unlabeled peptide with the sequence of rat Na1.2 IQ motif showed that apo CaM (a) was necessary and sufficient for binding, and (b) bound more favorably than calcium-saturated CaM. However, we could not monitor the Na1.2 residues directly, and no structure of full-length CaM (including the N-domain of CaM (CaM)) was determined. To distinguish contributions of CaM and CaM, we used solution NMR spectroscopy to assign the backbone resonances of a complex containing a C,N-labeled peptide with the sequence of human Na1.2 IQ motif (Na1.2) bound to apo C,N-CaM or apo C,N-CaM. Comparing the assignments of apo CaM in complex with Na1.2 to those of free apo CaM showed that residues within CaM were significantly perturbed, while residues within CaM were essentially unchanged. The chemical shifts of residues in Na1.2 and in the C-domain of CaM were nearly identical regardless of whether CaM was covalently linked to CaM. This suggests that CaM does not influence apo CaM binding to Na1.2.