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Analysis of a nicotine-related receptor binding site

Institution: University of California, San Francisco
Investigator(s): Laura England, Ph.D.
Award Cycle: 1997 (Cycle 6) Grant #: 6FT-0103 Award: $69,218
Subject Area: Nicotine Dependence
Award Type: Postdoctoral Fellowship Awards
Abstracts

Initial Award Abstract
Neurotransmitters are the primary chemical messengers that carry signals between nerve cells. Serotonin is one of the predominant neurotransmitters in the mammalian brain. Serotonin exerts its effects through interaction with a large number of functionally diverse receptor molecules. When serotonin binds to the receptor, the channel opens to allow sodium and potassium to flow into the nerve cell and initiate a neuronal response.

The study of this receptor is particularly relevant to the treatment of nicotine dependence because drugs that counter serotonin receptor activity have been demonstrated to reduce the rewarding effects of nicotine and to alleviate withdrawal symptoms. Also, this serotonin receptor is most closely related to the nicotinic receptor and provides a unique model system for the analysis of the structure and function of this group receptors.

The proposed research will investigate the structure of the site(s) to which serotonin and drugs bind to the type 3 serotonin receptor, with the aim of defining the interactions of the receptor with drugs at the molecular level. This investigation will provide the first high resolution picture of this important family of neurotransmitter receptors and will ultimately facilitate the rational design of selective drugs that modulate the serotonin receptor and other members of this family.

Final Report
We are investigating the structure of the site(s) to which serotonin and drugs bind to the type 3 serotonin receptor (5-HT3), with the aim of defining interactions of the receptor with drugs at the molecular level. The study of this receptor is relevant to the treatment of nicotine. dependence because drugs that inhibit serotonin receptor activity reduce the rewarding effects of nicotine and alleviate withdrawal symptoms. Also, the 5-HT3 receptor is closely related to the nicotinic receptors and has several experimental advantages for the analysis of the three-dimensional structure of this group of receptors. We are also investigating another receptor called vanilloid receptor type 1 (VRI). This rector is activated by capsaicin, the "hot" component of hot chili peppers as well as noxious heat and mo erately acidic conditions: This receptor is important because of its role in vascular control and pulmonary disease conditions. VR1 is proposed to play a role in the protective reactions from irritants such as smoke and nicotine. Its activity contributes to the symptoms of airway disease because it is potentiated by inflammatory mediators resulting. in an increased inflammatory response (neurogenic mflanunation). This study has three specific aims: 1) to use reactive versions of a novel peptide antagonist (a-conotoxin) to label the binding site of the 5-HT3 receptor and to map the interacting amino acid residues, 2) to produce the drug-binding domain of the 5-HT3 receptor to aid in the threedimensional structure determination of the drug-binding site, and 3) to investigate the mechanism of VRl potentiation in sensory neurons.

Toward the first aim, we have focussed on the development of small synthetic versions of the peptide antagonist (a-conotoxin). Synthetic versions of a-conotoxin will allow us to engineer peptide species which can react with the receptor, thereby allowing us to map the interacting amino acids. Our studies have determined some of the features of a-conotoxin that are important for its activity. This information will aid us in the design of reactive peptides. The small synthetic peptides we have generated to date have a low affinity interaction with the receptor and have limited utility for use in receptor cross-linking studies. We have synthesized full-length a-conotoxin but it does not have any 5HT 3 receptor antagonist activity presumably because it is not correctly folded during the synthesis. Toward the second aim, most expression strategies that we have analysed have yielded protein that does not bind drugs and is therefore not suitable for protein structure determination. With respect to the third aim, I have investigated many potential biochemical mechanisms whereby VRl may be modulated, as suggested by other groups' studies in sensory neurons. Our experiments suggest that the cellular lipid arachidonic acid may potentiate VRl activity under inflammatory conditions.

Our next step is to continue our efforts to produce full-length a-conotoxin and to fold it into its active conformation. Active peptides will then be reacted with the serotonin receptor and interacting amino acids will be identified. This will give us the first biochemical insight into how serotonin receptors interact with drugs. We will also continue our investigation of the effects of arachidonic acid on VRl potentiation. Since the potentiation of VR1 has been reported to be involved in neurogenic inflammation, the continuation of these studies may reveal new therapeutic targets for the management of airway disease.
Publications

Inactivation of a serotonin-gated ion channel by a novel polypeptide toxin from marine snails
Periodical: American Association for the Advancement of Science Index Medicus:
Authors: England LJ, Imperial J, Jacobsen R, et al ART
Yr: 1998 Vol: 281 Nbr: Abs: Pg: 575-578

Inactivation of a serotonin-gated ion channel by a novel polypeptide toxin from marine snails
Periodical: Science Index Medicus:
Authors: England LJ, Imperial J, Jacobsen R, et al ART
Yr: 1998 Vol: 281 Nbr: Abs: Pg: 575-578