Backbone Resonance Assignments of Complexes of Human Voltage-Dependent Sodium Channel NaV1.2 IQ Motif Peptide Bound to Apo Calmodulin and to the C-Domain Fragment of Apo Calmodulin

摘要

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 CaM_C were significantly perturbed, while residues within CaM_N were essentially unchanged. The chemical shifts of residues in Na_V1.2_IQp and in the C-domain of CaM were nearly identical regardless of whether CaM_N was covalently linked to CaM_C. This suggests that CaM_N does not influence apo CaM binding to Na_V1.2_IQp.