Structural Characterization of a Tarantula Toxin and its Interactions with Lipid Bilayers and Voltage-Gated Sodium Channels

Author

Krishnakoli Adhikary, CUNY Graduate CenterFollow

Date of Degree

9-2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Chemistry

Advisor

Sebastien Poget

Committee Members

Mandë Holford

Sharon M. Loverde

Subject Categories

Biochemistry, Biophysics, and Structural Biology

Keywords

ion channel, toxin, spider toxin, neurotoxin, tarantula toxin, sodium channel

Abstract

Voltage-gated ion channels are membrane protein complexes. They allow the selective flow of their respective ions down an electrochemical gradient through a central ion conduction pore surrounded by four voltage-sensing domains (VSDs). Various neurological and cardiovascular diseases are caused by mutations in voltage-gated ion channels (VGICs). One of the biggest challenges today is addiction to opioid medication. Coming up with non-opioid drug leads can be a real blessing for society. This is where voltage-gated sodium channel research comes in. Na_v1.7, Na_v1.8, and Na_v1.9 are responsible for pain sensations. Loss or gain of function in these channels can cause extreme pain or loss of sensation to pain.

Neurotoxins from venomous animals such as tarantulas, scorpions, snakes, cone snails, sea anemones, to name a few, are used to probe voltage-gated ion channels because they interact with these channels by either blocking the flow of ions by occluding the pore or modifying their gating properties, and cause changes in channel gating. So, these neurotoxins can be excellent drug leads for conditions arising due to defects in ion channels. For example, the FDA-approved non-opioid analgesic drug (used to treat chronic pain), Ziconotide (Prialt), is the synthetic version of a peptide neurotoxin (ω-MVIIA) from the venom of the marine snail Conus magus. This toxin regulates pain by selectively blocking N-type calcium channels on the surface of nerve cells.

In this study, we have characterized a peptide neurotoxin, GsAF2, from the venom of the Chilean rose tarantula, Grammostola rosea. These spider toxins form multiple disulfide bonds and fold into a knot through a motif called the inhibitor cysteine knot (ICK). This disulfide-rich, 31-residue peptide is known to have antiarrhythmic and analgesic properties — properties that make it a potentially useful drug candidate. Although the functional characteristics of the toxin have been studied extensively, its structural characteristics have remained a mystery. Understanding the structure-function relationship of the toxin is needed to understand its potential as a drug lead.

As a first step, we have heterologously expressed GsAF2 in an Escherichia coli (E. coli) - based system, through recombinant means to procure ample amounts of toxins efficiently and cost-effectively. We performed expression trials to find out the most optimal route for expressing, purifying, cleaving, and refolding the toxin into its native fold. Once the recombinant toxin was obtained, we successfully co-eluted it with the native toxin and tested its bioactivity using automated patch clamp electrophysiology against Na_v1.7 to establish that the toxin was correctly folded.

Next, we expressed ¹⁵N- and ¹³C-labeled toxin and performed two and three-dimensional NMR experiments to determine its structure in solution. The structure of the toxin was similar to other spider toxins, with an amphipathic surface. Our structure had a flexible loop as seen in some other spider toxins.

Finally, we proceeded to characterize the toxin in bicelles in the presence and absence of the voltage-sensing domain from a bacterial sodium channel, NaChBac. NaChBac acts like a simple model for studying eukaryotic sodium channels. Voltage-gated ion channels are embedded in lipid membranes. Toxins interacting with these channels generally anchor themselves in the membrane to reach the channel. Hence, understanding the lipid-binding characteristics of a toxin is essential to understanding its interaction with voltage-gated ion channels. We performed ¹⁵N-HSQC experiments in bicelles and in the presence of the voltage-sensing domain. We also performed paramagnetic relaxation enhancement experiments to understand which residues of the toxin resided near the lipid interface. Our results indicate that two of the tryptophan residues in the toxin reside near the membrane interface, and another one helps pull the toxin into the hydrophobic core to interact with the voltage-sensing domain of NaChBac. These results can be further exploited in the future to understand the binding characteristics of the toxin even better.

Overall, we have solved the structure of a tarantula toxin and investigated its residues important for its functionality at an atomic level. This project is a steppingstone to further the knowledge of this analgesic peptide and provides an excellent opportunity for future researchers to understand the mechanism of action of peptide neurotoxins on voltage-gated ion channels.

Recommended Citation

Adhikary, Krishnakoli, "Structural Characterization of a Tarantula Toxin and its Interactions with Lipid Bilayers and Voltage-Gated Sodium Channels" (2025). CUNY Academic Works.
https://academicworks.cuny.edu/gc_etds/6496