A Unique Binding Domain in NAV1.7 Accounts for Its Selective Regulation by CRMP2

Abstract

Treatment of chronic pain remains a major challenge for modern medicine and few treatments are both safe and effective. Here, using a peptide microarray, a 15 amino acid regulatory sequence unique to NaV1.7 that is essential for its function was discovered, and was named the NaV1.7-CRMP2 regulatory sequence (NaV1.7-CRS). This unique domain serves as the binding interface for a crucial NaV1.7 regulatory protein, the cytosolic phosphoprotein collapsin response mediator protein 2 (CRMP2), and binding between the NaV1.7-CRS and CRMP2 is involved in neuropathic pain. CRMP2 bound to the NaV1.7-CRS with low micromolar (<1 µM) affinity as determined using microscale thermophoresis (MST). Importantly, when this region is lost, tetrodotoxin sensitive currents in primary sensory neurons are approximately halved without affecting tetrodotoxin resistant currents. Furthermore, treatment with a membrane delimited cell penetrant interfering peptide (Myr-TAT-NaV1.7) reduced NaV1.7 currents in rat primary sensory neurons and reversed mechanical allodynia in a rat spared nerve injury model of neuropathic pain. The findings presented here demonstrate that this peptide exerts its effects by altering NaV1.7 trafficking, resulting in decreased presynaptic NaV1.7 localization and reduced evoked spinal CGRP release. Interfering with NaV1.7-CRMP2 coupling does not result in unwanted side effects, such as motor impairment, and does not affect physiological thermal nociception. Furthermore, adeno-associated virus serotype 9 (AAV9) mediated delivery of a peptide encoding the CRS sequence reduces NaV1.7 currents and relieves mechanical allodynia, which provides proof-of-concept for NaV1.7 targeted gene therapy. In primary DRG sensory neurons isolated from nonhuman primates, interfering with NaV1.7 and CRMP2 coupling attenuated whole cell NaV1.7 currents. This finding highlights the translational potential of this domain-targeted strategy. Collectively, these results support the conclusion that the NaV1.7-CRS is a novel pharmacological site that can serve as the foundation for development of therapeutics by indirectly interfering with the regulation of NaV1.7.