The Structural Characterization of the Human Cardiac Sodium Channel Voltage-Sensor Domain IV Paddle Motif

Abstract

Voltage-gated sodium channels (VGSCs) are membrane proteins that serve important functions in the central and peripheral nervous systems (C/PNS) and cardiac and skeletal muscle. Diseases that can be caused by malfunctioning VGSCs (often due to modified gating properties) include epilepsy, chronic pain, and several ailments afflicting the heart. Interestingly, peptide toxins from venomous and poisonous animals have been known to target VGSCs. Site 3 and 4 peptide neurotoxins are known to interact with the voltage-sensing domains of VGSCs, modifying their gating properties. It has been proposed that peptide neurotoxins may be used directly or as lead compounds for rational drug design to alleviate symptoms caused by channelopathies. An example of this approach is the drug Prialt, an ω-conotoxin, used to alleviate intractable pain in patients. By probing the structural details of the interaction between peptide neurotoxins and VGSCs, we will gain more insight into the function of these toxins and what governs their strong and specific effect. VGSCs are large, highly hydrophobic and heavily post-translationally modified proteins, thus making it unlikely that X-ray crystallography, solution-state NMR or cryo-EM will be successful in producing structural information on intact protein at the atomic level. As an alternative strategy, we are studying the main binding sequence for site 3 and 4 toxins, the S3b-S4 paddle motif, in complex with an interacting gating modifier toxin of the VGSC NaV1.5. VSD IV of NaV1.5 is the known target of many site-3 α-scorpion and sea anemone toxins that inhibit fast inactivation of the channel. We have chemically and biosynthetically synthesized the 37 residue paddle motif peptide and characterized it through circular dichroism spectroscopy, MALDI-TOF mass spectrometry, and solution state NMR, producing the first backbone assignments of a mammalian VGSC paddle motif.