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Structural determinants of alpha-toxin: nAChR interactions

Institution: University of California, San Diego
Investigator(s): Siobhan Malany, Ph.D.
Award Cycle: 1998 (Cycle 7) Grant #: 7FT-0085 Award: $70,000
Subject Area: Nicotine Dependence
Award Type: Postdoctoral Fellowship Awards

Initial Award Abstract
Information from the brain is translated into an electrical response within the central nrevous system by the action of chemical compunds (neurotransmitters) binding two sites on tissue membranes (receptors). The focus of our research is one of these receptors, namely the nicotinic acetylcholine receptor, because it binds nicotine. The receptor consists of five subunits which are arranged around a funnel-shaped channel. When two molecules of neurotransmitter bind to the subunits at the same time, the channel opens to allow the passage of charged particles (ions) which leads to message propagation. The nicotinic receptor is the best studied of several related and medically important receptors. However, our knowledge of the three-dimensional structure of this model receptor and how the receptor-drug complex causes its effects is limited.

We address the structure-function question by analyzing the interaction of a muscle receptor with a compound which is highly specific for this receptor. The compound is a protein component of snake venom which is a neurotoxin because the protein binds irreversibly to the receptor and inactivates it. The advantage of this compound is that we know its configurations.

The goal of our research is to provide a comprehensive view of how the toxin and the channel interact. We will do this by determining the important regions for toxin-receptor interaction by looking where the toxin and receptor bind together. Since we know the structure of the toxin, we can determine the structure of the receptor at those sites. It is important to increase our understanding of the relationship between the structure and the function for toxin or drug interactions with these types of receptors because they will provide insight into how the receptor channels are important to memory and learning events and to neurological disorders which interfere with these events.

Final Report
Nicotine dependence arises from the interaction of nicotine with a macromolecule in the brain termed the nicotinic acetylcholine receptor (nAChR). Binding of nicotine or the physiological neurotransmitter, acetylcholine activates the receptor. Subsequently, the receptor opens to allow charged particles like calcium and sodium ions to pass through, thus acting as a channel for the propagation of electrical information from the brain to the various tissues. One of the approaches to understanding how nicotine, and ligands in general, interacts with its target is to reveal the structural elements of the target responsible for binding specificity. Of the family of ligand-gated ion channels, the nicotinic acetylcholine receptor from skeletal muscle is the best characterized but still a high-resolution structure is lacking. Our research seeks to identify key components of the nAChR involved in binding ligands and to delineate the relative spatial arrangement of these components at the binding interfaces. The nAChR is composed of four subunits arranged in a rosette pattern aya8(3. In adult muscle receptors, the y subunit is replaced by s. Ligands bind at interfaces between the aS and ay (as) subunit pairs. Our studies utilize an a-neurotoxin of known structure isolated from snake venom of the cobra Naja mossambica mossambica (NmmI). Unlike acetylcholine or nicotine, Nmml inactivates the receptor, thus acting as a paralysis-inducing toxin. NmmI serves as a unique probe in our efforts to map the subunit binding interfaces of the nAChR because of its high selectivity for the ay subunit interface over that formed by aE. In addition, the a-neurotoxin which contains three finger-like loops, contacts a large surface area of the receptor.

Our research has involved generating a series of amino acid mutations in both the a-neurotoxin and the receptor, expressing the two proteins from their mutant DNA, conducting binding assays, and measuring the alteration in binding of the complex due to the introduced mutations. Our results have revealed specific amino acid residues in both the a subunit and the y subunit of the receptor governing Nmml cc-toxin recognition. Similarly, we have analyzed residues in the three loops of the a-neurotoxin critical for binding to the aS and ay subunit interfaces. We further examined charged substitutions on both the a (or y) subunit of nAChR and Nmml and analyzed the energetics of binding for the combination of double mutations by a method called thermodynamic mutant cycle analysis. Through this method, we have delineated pairwise-residue interactions that stabilize the high affinity NmmI-nAChR complex. Moreover, our results have revealed that residues on the middle loop and on the concave face of NmmI serves as an anchor of contact to both the a and y subunits. A refined picture has emerged of how Nmml orients with respect to the a and y interfaces n+ +i,o ~„ gao „f- +l;A „ e CHu.

Subunit interface selectivity of the alpha-neurotoxins for the nicotinic acetylcholine receptor
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Osaka H, Malany S, Kanter JR, Sine SM, Taylor P ART
Yr: 1999 Vol: 274 Nbr: Abs: Pg: 9581-9586

Toxins selective for subunit interfaces as probes of nicotinic acetylcholine receptor structure
Periodical: Journal de Physiologie (Paris) Index Medicus:
Authors: Taylor P, Osaka H, Molles BE, et al ART
Yr: 1998 Vol: 92 Nbr: Abs: Pg: 79-83

Complementary binding studies between alpha-neurotoxin and the nicotinic acetylcholine receptor
Periodical: Journal de Physiologie (Paris) Index Medicus:
Authors: Malany S, Ackermann E, Osaka H, Taylor P ABS
Yr: 1998 Vol: 92 Nbr: Abs: 462-463 Pg:

Pairwise electrostatic interactions between alpha-neurotoxins and delta, epsilon and epsilon subunits of the nicotinic acetylcholine receptor
Periodical: Journal of Biological Chemistry Index Medicus:
Authors: Osaka H, Malany S, Molles BE, Sine SM, Taylor P ART
Yr: 2000 Vol: 27 Nbr: Abs: Pg: 5478-5484

Alpha-neurotoxin positioning at the subunit interfaces of the nicotinic acetylcholine receptor
Periodical: FASEB Journal Index Medicus:
Authors: Malany S, Osaka H, Taylor P ABS
Yr: 2000 Vol: 14 Nbr: A1359 Abs: Pg:

Subunit selective toxins as probes of nicotinic acetylcholine receptor structure
Periodical: Pflugers Archiv. European Journal of Physiology Index Medicus:
Authors: Taylor P, Malany S, Molles BE, Osaka H, Tsigelny I ART
Yr: 2000 Vol: Nbr: Abs: Pg: