Nicotinic acetylcholine receptors of neuronal tissue are less well characterized than the corresponding nicotinic receptors of skeletal muscle. Neuronal tissue is anatomically much more complex than that of skeletal muscle, and in order to understand the physiology and biochemistry of receptors in neuronal tissue, suitable probes must be developed. The recently discovered K-neurotoxins are the only known substances capable of detecting and distinguishing neuronal acetylcholine receptors. The long term significance of this research lies in the potential use of K- neurotoxins in characterizing neuronal nicotinic receptors in a variety of disease states related to cholinergic function in the brain and in the development of drugs which can distinguish subtypes of neuronal receptors. Both presenile and senile forms of Alzheimer's disease as well as Huntington's disease appear to involve deficits in cholinergic neuron function in the central nervous system. Also, studies of certain epilepsy patients suggest that an autoimmune disease against the neuronal nicotinic receptor may exist which would be analogous to the attack on the muscle receptor seen in Myasthenia Gravis. Our understanding of these processes should be facilitated as more information is gathered concerning the biochemical and physiological properties of neuronal receptors. If specific receptors could be activated or blocked without interfering with other receptor, their functions could be determined much more precisely and much more precise targeting of drug effects could be evaluated. The long range goal of this research project is to develop the K-neurotoxins as tools for investigating the various classes and sub-classes of nicotinic acetylcholine receptors and related proteins. The immediate focus of this research will be in developing systems for the production and mutagenesis of the K-neurotoxins, and to investigate the relationship between structure and function in this unique family of neurotoxins and their interactions with acetylcholine receptor subunits.
The Specific Aims of this Research Project are 1) to efficiently express a gene coding for a K-neurotoxin in a procaryotic system by chemically synthesizing that gene and placing it in a bacterial expression construct, 2) to isolate significant qualities of the neurotoxin for use in tertiary structure investigations, 3) to their interactions with acetylcholine receptors by site specific mutagenesis, and 4) to determine the three-dimensional structure of a K-neurotoxin by x-ray crystallography.
| Dewan, J C; Grant, G A; Sacchettini, J C (1994) Crystal structure of kappa-bungarotoxin at 2.3-A resolution. Biochemistry 33:13147-54 |
| Fiordalisi, J J; al-Rabiee, R; Chiappinelli, V A et al. (1994) Affinity of native kappa-bungarotoxin and site-directed mutants for the muscle nicotinic acetylcholine receptor. Biochemistry 33:12962-7 |
| Fiordalisi, J J; Grant, G A (1993) Evidence for a fast-exchange conformational process in alpha-bungarotoxin. Toxicon 31:767-75 |
| Sachettini, J C; Patel, S; Scapin, G et al. (1992) Crystallization of kappa-bungarotoxin: preliminary X-ray data obtained from the venom-derived protein. J Mol Biol 226:559-62 |
| Fiordalisi, J J; Fetter, C H; TenHarmsel, A et al. (1991) Synthesis and expression in Escherichia coli of a gene for kappa-bungarotoxin. Biochemistry 30:10337-43 |
