Research Summary

The nicotinic acetylcholine receptor (nAChR) mediates synaptic transmission at the neuromuscular junction, in peripheral autonomic ganglia, and in the central nervous system. The nAChR belongs to a super-family of ligand-gated channels which include the glycine and GABA-A receptors. In the case of the nAChR, receptor activation occurs after binding of the physiological agonist, acetylcholine (ACh), to the receptor complex leading to the transient formation of a cation-selective channel. Antagonists with relative specificity for the nAChR include tubocurarine and the snake venom-derived neurotoxins such as alpha-bungarotoxin (BGTX) and alpha-cobratoxin. Our major interest is in determining the molecular and structural basis for the specificity of drug-receptor interactions. To accomplish this we are carrying out protein and peptide biophysical studies of receptor fragments. These studies include determining the solution structure of toxin-receptor fragment complexes using modern multidimensional NMR spectroscopic techniques. We also are applying molecular biological approaches to this problem by using heterologous expression and site-directed mutagenesis of both receptor and toxin residues in order to elucidate the structure-function relationship in this system. Both approaches are complementary and will facilitate the complete structural determination of the ligand binding site in the nicotinic acetylcholine receptor. Such information could provide the basis for the rational design of drugs selective for such sites.

More recent efforts are directed to exploiting the binding determinants responsible for BGTX action by transplanting these into other subunits normally insensitive to BGTX.  We have termed the peptide sequence with this property a “pharmatope”.  We find that I some cases BGTX binding to such a pharmatope produces functional blockade of receptor action through an allosteric as opposed to a competitive mechanism.  One further advantage to a pharmatope-tagged receptor subunit is that the many commercially available derivates of BGTX can now be used to label and functionally inhibit receptor complexes containing the tagged subunit.  We are now pushing this approach to the organismic level by preparing a mouse using the homologous recombination technique (i.e., knock-in) in which the alpha3 gene is replaced by one containing a five amino substitution rendering the mutant subunit sensitive to BGTX whereas the wild-type alpha3 is completely insensitive to BGTX.  This mouse was the pioneer mouse generated in the Brown University Transgenic Facility is collaboration with Dr. Jan Klysik.  The heterozygous mice are completely normal and breed well.  We are currently assessing the functional sensitivity of the neuronal nicotinic receptors in the peripheral ganglia from these knock-in mice.  The aim here is to use these mice to explore the physiological function of the alpha3 subunit in the central nervous system utilizing a standard pharmacological dissection approach.

Publications

Sanders, T. and Hawrot, E. (2004) A novel pharmatope tag inserted into the beta4 subunit confers allosteric modulation to neuronal nicotinic receptors. Journal of Biological Chemistry; 279:51460-5. http://www.jbc.org/cgi/content/full/279/49/51460

Zeng, H. and Hawrot, E.  (2002) NMR-based binding screen and structural analysis of the complex formed between alpha-cobratoxin and an 18-mer cognate peptide derived from the alpha1 subunit of the nicotinic acetylcholine receptor from Torpedo californica.  Journal of Biological Chemistry 277:37439-37445. http://www.jbc.org/cgi/content/full/277/40/37439

Moise, L., Zeng, H., Caffery, P., Rogowski, R.S., and Hawrot, E. (2002) Structure and function of alpha-bungarotoxin. Journal of Toxicology-Toxin Reviews, 21(3):293-317. http://search.epnet.com/direct.asp?an=7705507&db=aph

Moise, L., Piserchio, A., Basus, V.J., Hawrot, E. (2002) NMR structural analysis of alpha-bungarotoxin and its complex with the principal alpha-neurotoxin binding sequence on the alpha7 subunit of a neuronal nicotinic acetylcholine receptor. Journal of Biological Chemistry 277:12406-17. http://www.jbc.org/cgi/content/full/277/14/12406

Zeng, H., Moise, L., Grant, M.A., & Hawrot, E. (2001) The solution structure of the complex formed between alpha-bungarotoxin and an 18mer cognate peptide derived from the alpha1 subunit of the nicotinic acetylcholine receptor from Torpedo californica. Journal of Biological Chemistry, 276:22930-40. (http://www.jbc.org/cgi/content/full/276/25/22930)

Spura, A., Riel, R., Freedman, N.D., Agrawal, S., Seto, C., & Hawrot, E. (2000) Biotinylation of substituted cysteines in the nicotinic acetylcholine receptor reveals distinct binding modes for alpha-bungarotoxin and Erabutoxin a. Journal of Biological Chemistry 275: 22452-22460. (http://www.jbc.org/cgi/content/full/275/29/22452)

Levandoski, M.M., Caffery, P., Rogowski, R.S., Shi, Q.-L., & Hawrot, E. (2000) Recombinant expression of alpha-bungarotoxin in Pichia pastoris facilitates identification of mutant toxins engineered to recognize neuronal nicotinic acetylcholine receptors. The Journal of Neurochemistry, 74: 1279-1289.

Blein, S., Hawrot, E., & Barlow, P. (2000) The metabotropic GABA receptor: molecular insights and their functional consequences. Cell and Molecular Life Science, 57: 635-650.

Spura, A., Russin, T.S., Freedman, N., Grant, M., McLaughlin, J.T., & Hawrot, E. (1999) Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the alpha-bungarotoxin binding site. Biochemistry 38: 4912-4921.

Rosenthal, J.A., Levandoski, M.M., Chang, B., Potts, J.F., Shi, Q.-L., and Hawrot, E. (1999) The functional role of positively charged amino acid side chains in alpha-bungarotoxin revealed by site-directed mutagenesis of a His-tagged recombinant alpha-bungarotoxin. Biochemistry 38: 7847-7855.

Grant, M.A., Gentile, L.N., Shi, Q.-L., Pellegrini, M., and Hawrot, E. (1999) Expression and spectroscopic analysis of soluble nicotinic acetylcholine receptor fragments derived from the extracellular domain of the alpha-subunit. Biochemistry 38: 10730-10742.

Levandoski, M.M., Lin, Y., Moise, L., McLaughlin, J.T., Cooper, E., & Hawrot, E. (1999) Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to alpha-bungarotoxin. Journal of Biological Chemistry 274: 26113-26119.

Hawrot, E., Xiao, Y., Shi, Q.-L., Norman, D., Kirkitadze, M., and Barlow, P. (1998) Demonstration of a tandem pair of complement protein modules in GABA-B receptor 1a. FEBS Letters 432: 103-108.

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