Structure and Mechanism of a Novel Scorpion Peptide that Inhibits Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signal Transmission
Ashlee Rowe
Assistant Professor, University of Oklahoma
Voltage-gated sodium channels Nav1.7 and Nav1.8 regulate pain signal transmission in sensory neurons. Arizona bark scorpions produce peptide toxins that activate Nav1.7, which then recruits Nav1.8 to initiate pain signals. However, when predatory grasshopper mice are stung, peptides act as analgesics by inhibiting Nav1.8 and blocking pain signals. The biophysical interactions between inhibitory peptides and Nav1.8 provide a system for linking mechanisms of altered pain-signal transmission with pain-resistance phenotypes. To determine the mechanisms underlying peptide-mediated pain resistance, we tested synthetic bark scorpion venom peptides on a recombinant grasshopper mouse Nav1.8 channel (OtNaV1.8) and identified a novel peptide, NaTx36, that inhibits OtNav1.8 in a concentration dependent manner. NaTx36 hyperpolarizes OtNav1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis demonstrated that the first gating charge arginine in the domain I (DI) S4 voltage sensor and an acidic glutamate in the DII SS2 – S6 pore loop are critical for the inhibitory effects of NaTx36. Computational models revealed that while a DI S1 – S2 asparagine stabilizes the NaTx36 – OtNav1.8 complex, residues in the DI S3 – S4 linker enable a toxin glutamine to bind the DI voltage sensor S4 first gating charge. Electrophysiological analyses suggest that NaTx36 traps the DI voltage sensor in the activated position. Surprisingly, the models predicted that NaTx36 binds residues in the DII S5 – SS1 loop, not the DII SS2 – S6 glutamate. The models suggest that the DII SS2 – S6 glutamate allosterically alters the conformation of the DII S5 – SS1 loop, enhancing interactions with NaTx36. Nav1.8 is critical for pain signal transmission. While the structural components that form the voltage sensors, and activation/inactivation gates are known, mechanisms that couple activation to inactivation are not completely understood. Thus, NaTx36 and OtNav1.8 provide a toolkit for investigating the relationship between activation and inactivation, and how variation in that relationship alters pain phenotypes.
I am a neurobiologist in the Department of Biology and Graduate Program in Cellular & Behavioral Neurobiology at the University of Oklahoma. My research program integrates venom biochemistry with genetics, sensory physiology, and animal behavior. Because ion channels are crucial for all physiological processes, they are targets of venom-derived neurotoxins produced by diverse taxa. I use scorpion peptide toxins that reversibly modify the activation and inactivation gates of voltage-dependent sodium and potassium ion channels to probe these targets in the nerve and muscle tissue of predatory mice. Interactions between venom peptides and target channels enable the examination of the molecular mechanisms by which animals transduce sensory stimuli, interpret signals, and generate motor responses. The strength of this system is that differential sensory phenotypes (e.g., inter-individual variation in pain sensitivity) expressed in natural populations can be linked to genetic variation in ion-channel encoding genes, laying the foundation for contributions to translational medicine. I am currently collaborating with proteomics and structural biology researchers to elucidate the biophysical mechanisms of peptide-mediated inhibition of the pain-regulating sodium channel Nav1.8.