Date of Degree

9-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor(s)

Sébastien F. Poget

Committee Members

Fred Naider

Mandë Holford

Ming Tang

Thomas V. McDonald

Subject Categories

Biochemistry | Biophysics | Molecular Biology | Structural Biology

Keywords

Peptide Neurotoxin, Nuclear Magnetic Resonance Spectroscopy, Voltage-gated sodium channel, Voltage-sensor domain, Ion channel, toxin

Abstract

In nature, peptide toxins are an abundant resource, produced both by marine and terrestrial organisms. A major target of these peptide toxins is the group of the highly important voltage-gated ion channels. Due to their high specificity and affinity, peptide toxins have been used for over a decade in discovery and characterization of voltage-gated ion channels. Although peptide toxins have been extensively characterized structurally, the structural characterization of eukaryotic voltage-gated sodium channels has seen much less progress, due to their large size and high hydrophobicity. Voltage-gated sodium channels play crucial roles in many physiological processes, and when these processes are disrupted, due to malfunctioning ion channels, they lead to diseased states known as channelopathies. It is believed that some channelopathies can be corrected through fine tuning of the gating properties of the diseased channels through the use of drugs or peptide toxins. If the structural interactions of these peptide toxins with their cognate voltage-gated sodium channels can be unraveled, we may be able to use them as drugs directly or as lead compounds for the thoughtful design of small molecules that exploit these interactions. Here, I have taken the following steps towards an increased structural understanding of ion channel-toxin interactions:

Firstly, we have shed light on the first high-resolution structure of a novel class of peptide toxins known as teretoxins with the determination of the structure of Tv1. Tv1 was isolated from the terebrid marine snail Terebra variegata in nanogram quantities and its sequence determined by direct mass spectrometry sequencing. The peptide was chemically synthesized and refolded, and then subjected to solution-state NMR experiments. We were able to solve the structure of Tv1 to a resolution 0.74 Å using 2D-homonuclear/heteronuclear NMR techniques. Although Tv1’s target remains elusive, structural features suggest that its natural target is an ion channel.

Voltage-sensor domains of voltage-gated ion channels are responsible for sensing changes in the membrane potential of excitable cells and translating this information into the various states of the pore (open, closed, inactivated), but the mechanism by which these states are transferred from the voltage-sensor domain to the channel remains unknown. Here we have expressed a voltage-sensor domain of the human cardiac voltage-gated sodium channel. We have demonstrated the ability to isotopically label the voltage-sensor domain in a bacterial expression system. 15N-labeled voltage-sensor domain was subjected to 15N-TROSY experiments to demonstrate the feasibility of structural studies. The Voltage-Sensor Domain under study produced little to no signal in n-DPC micelles, but marked improvement was obtained in a bicelle system. In spite of such improvement, system conformational heterogeneity seems to be apparent. We were also able to pull down a toxin from the venom of the tarantula Grammastola rosea using immobilized voltage-sensor domain, with the potential establishment of a method for the discovery of novel site 3 toxins by pull down with NaV1.5.

Paddle motifs are the target of many gating-modifier toxins and have been shown to bind toxins independent of their background (placement into non-native channels) and in isolation. Here we present the synthesis (both chemical and biological) and characterization of a mammalian 37 residue NaV paddle motif. We subjected an isotopically labeled paddle motif of DIV of the human cardiac voltage-gated sodium channel (NaV1.5) to various 2D and 3D NMR experiments and assigned 86% of the backbone residues. We also recorded NMR spectra of the paddle motif in the presence of ApA, a known site-3 gating modifier toxin, which yielded complete loss of NMR signal, indicating that an interaction is occurring. Through the establishment of our backbone assignments, interactions of known site 3 gating-modifier toxins and NaV1.5 DIV S3-S4 paddle motif can be characterized, yielding mechanistic insights and knowledge that could be used in drug design.

Peptide toxins and voltage-gated ion channels have been used to unravel the secrets of each other for years, and our work continues in that direction. With Tv1 we have shown the ability to structurally characterize a peptide toxin using limited resources, which will open the door to structurally characterizing toxins thought to be restricted due to sample size limitations. With the NaV1.5 VSD and paddle motif of DIV we have presented a strategy to express and structurally characterize transmembrane fragments of a eukaryotic voltage-gated sodium channel in the absence and presence of gating-modifier toxins by NMR spectroscopy. Taken together, the experiments we conducted have increased the atomic level knowledge of toxin and eukaryotic voltage-gated ion channel biology.

 
 

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