NMR solution structure and analysis of isolated S3b-S4a motif of repeat IV of the human cardiac sodium channel

Voltage-gated sodium channels are membrane proteins that play an important role in the propagation of electrical signals by mediating the rising phase of an action potential. Numerous diseases, including epilepsy, extreme pain, and certain cardiac arrhythmias have been linked to defects in these channels. The S3b-S4a helix-turn-helix motif (paddle motif) is a region of the channel that is involved in voltage sensing and undergoes significant structural changes during gating. It is also the binding site for many gating-modifier toxins. We determined the solution structure of the paddle motif from the fourth repeat of NaV1.5 in dodecylphosphocholine micelles by NMR spectroscopy and investigated its dynamics and micelle interactions. The structure displays a helix hairpin with a short connecting loop, and likely represents the activated conformation with three of the first four gating charges facing away from S3. Furthermore, paramagnetic relaxation measurements show that the paddle motif is mainly interacting with the interface region of the micelle. NMR relaxation studies reveal that the paddle motif is mostly rigid, with some residues around the loop region and the last 4 residues on the C-terminus displaying heightened mobility. The structural findings reported here allowed the interpretation of three disease-causing mutations in this region of the human cardiac sodium channel, S1609W, F1617del and T1620M. The establishment of this model system for NMR studies of the paddle region offers a promising platform for future toxin interaction studies in the cardiac sodium channels, and similar approaches may be applied to other sodium channel isoforms.