Allosteric regulation of voltage-gated sodium channels by lipophilic neurotoxins

Voltage-gated sodium channels (NaVs) are large, transmembrane proteins critical for bioelectrical signaling. NaVs permit sodium ion influx into cells by opening in response to membrane depolarization, a process involving large protein conformational changes over millisecond response times. Channel dysregulation is associated with a number of human pathologies, including chronic pain, epilepsies, and arrhythmias. Therefore, efforts to better understand NaV dynamics and to rationally design allosteric modulators of channels are of considerable scientific interest. NaVs are the targets of numerous natural product neurotoxins, including batrachotoxin (BTX), found in the skin secretions of poison dart frogs, which function as allosteric ligands. A full channel agonist, BTX eliminates NaV inactivation and shifts the voltage-dependence of channel activation, among other effects, making it a privileged tool for biophysical studies of NaVs. We have utilized BTX and related toxins to provide insight into the mechanisms by which these toxins modulate NaVs and to better understand how small molecules can influence NaV dynamics. Herein, we describe efforts to elucidate the molecular determinants of the BTX binding site as well as to advance our understanding of toxin structure-function relationships. Aided by computational docking, we propose a novel binding pose for BTX in the central pore of the channel, distal from the canonical local anesthetic binding site. Our studies have led to the identification of multiple amino acid residues in the NaV inner pore that discriminate between BTX and its mirror-image enantiomer. Through this work, we have identified the first known mutations in NaV that increase BTX affinity. Our efforts additionally identify a single point mutation in NaV that seemingly decouples toxin binding from its functional effects on the channel. Finally, studies in collaboration with Dr. Tim MacKenzie have produced a novel toxin derivative, BTX-yne, that eliminates fast inactivation in NaV while permitting slow inactivation. Use of this toxin derivative has enabled direct measurement of slow inactivation in both wild-type and mutant channels, advancing our understanding of this poorly-understood mechanism. Our studies of BTX have been done in parallel with analogous experiments using the neurotoxin, veratridine (VTD). We show VTD, a compound with a binding site that overlaps that of BTX, has disparate functional effects on NaV, dependent on the toxin equilibration protocol. We demonstrate this Janus-faced behavior is not isoform-specific and provide evidence that the characteristic tail current observed in VTD-agonized channels arises from so-called window current. Finally, we expand our understanding of the effects of BTX to include the influence of this toxin on cultured neuronal cells. In these experiments, we find that BTX induces hyperexcitability at concentrations two orders of magnitude below the measured EC50. We provide preliminary evidence that BTX has membrane protein targets beyond NaV with experiments studying voltage-gated calcium channels in neurons. These results provide insight into the profound lethality of this toxin and serve as a foundation for future experiments to examine the influence of BTX, VTD, and related toxins on neuronal cell activity and action potentials

Keywords: Chemistry.
Advisor: Du Bois Justin
Cegelski Lynette
Khosla Chaitan