We pursue biochemical and pharmacological studies aimed at understanding the fundamental structure-function relationship of nicotinic acetylcholine receptors (nAChRs). We also study the molecular basis for the highly specific recognition of muscle-type nAChRs by certain snake venom-derived toxins classified as alpha-neurotoxins. More recently, we have used homologous recombination techniques to construct a knock-in mouse in which the alpha3 gene encoding one subtype of neuronal nAChRs has been minimally mutated to impart pharmacological sensitivity to the classic nicotinic antagonist, alpha-bungarotoxin. These mice should enable a systematic determination of the role of alpha3-containing nAChRs in behavior and nervous system function.

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 in 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 in 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.

Many drugs—both those that are therapeutically useful and those that are not—physically interact with receptor proteins found on the cell surface of neurons and other excitable tissue. I am interested in determining the molecular and structural basis for the remarkable specificity of drug-receptor interactions in the brain. In particular, I am interested in the nicotinic acetylcholine receptor, which mediates synaptic transmission at the junction between motor nerves and skeletal muscle. The function of the neuromuscular junction can be completely blocked by alpha-neurotoxins derived from snake venom that work by binding to the receptor. The specificity and potency of the alpha-neurotoxins found in snake venom are so great that they represent an ideal toward which drug developers and medicinal chemists strive. The ideal is a drug extremely active at low concentrations that does not interact with irrelevant receptors, thus reducing the likelihood of side effects. Working toward that ideal, and to increase our understanding of how receptors work, I am pursuing a number of biophysical studies with other researchers involving receptor fragments. With more detailed structural information describing the toxin-receptor interface, it should be possible for us to engineer the toxin by mutagenesis so as to alter its specificity.

I became interested in this line of research as a result of my interests in chemistry and in the functioning of the brain. Both of these areas of interest were satisfied by studies of drugs and toxins and their interactions with receptors important for brain and nervous system function.

2010-2011 NIH/NCRR (1S10RR027027-01) Acquisition of an LTQ Orbitrap Velos ETD Mass Spectrometer

2010-2015 NIH (1T32GM077995-01A2) Predoctoral Training Program in Trans-Disciplinary Pharmacological Sciences

2010-2012 National Science Foundation (EPS-1005789) High-Capacity Cyber-Connectivity to the Jewelry District Campuses in Providence, RI

2010-2017 National Science Foundation (EPS-10040507; subaward to Brown University from URI) Rhode Island Experimental Program to Stimulate Competitive Research (RI-EPSCoR): Infrastructure to Advance Life Sciences in the Ocean State

2011-2015 NIH/NIA (1R21AG038774-01A1) Effects of Alzheimer's Disease on Hippocampal alpha7-nAChR Protein Interactors

2015-  NIH/NHLBI (1RO1HL128661-01; PI:Choudhary, Gaurav) Nicotinic Acetylcholine Receptors and RV Dysfunction in Pulmonary Hypertension

2005-2009 NIH (CSR) BSCT/BPNS Study Section (Biophysics of Neural Systems)
2010 NIH (CSR) ZRG1 MDCN-B(02) Special Emphasis Panel
2011 NIH (CSR) MNPS Study Section (ad hoc member)
2013 NIH (CSR) NINDS SNRP Study Section (ad hoc)

2014 NIH (CSR) STOD Study Section

2015 NIH (CSR) CNNT Study Section (ad hoc)

2011 Fellow of the American Association for the Advancement of Science (AAAS)

2001-2006 Editorial Board, Journal of Biological Chemistry

Member, American Association for the Advancement of Science, American Society for Pharmacology & Experimental Therapeutics, American Society for Biochemistry and Molecular Biology, Society for Neuroscience, Association of Medical School Pharmacology Chairs