The three fingered alpha-neurotoxin from the venom of Naja mossambica mossambica (Nmml) has been a valuable tool in our continuing effort to identify residues that contribute to toxin binding and in determining the relative orientation of the bound toxin. This is due to the toxin's differing affinities for the agonist binding sites formed at the alpha-gamma, alpha-delta, and alpha-epsilon subunit interfaces of the nicotinic acetyicholine receptor with approximately three orders of magnitude lower binding affinity for the alpha-epsilon interface versus the alpha-gamma or alpha-delta interfaces (Kd of 100 nM versus 100 pM, respectively). Despite having comp arable KD) values and considerable homology, binding of Nmml appears to be dictated by electrostatic interactions at the on subunit interface while recent mutagenic studies indicate that this is not the case at the alpha-gamma subunit interface. To delineate this paradox, I will exploit Nmml's unique binding profile by constructing nicotinic acetyicholine receptors using epsilon rather than gamma subunits. They will be assembled in a stoichiometric relationship of alpha-2-beta-epsilon-delta which corresponds to the subtype found in adult muscle tissue. This will allow us to examine the effects of residue replacement on toxin binding at the alpha-delta subunit interface without concern that our observations are being affected by inter-actions of the toxin with the other binding domain. In the second portion of our study we will develop soluble model of the receptor-toxin complex that can be used for spectroscopic and crystallographic study. This will be accomplished by transfecting appropriately truncated cDNA of the receptor subunits into an expression system followed by isolation purification. Accomplishment of these goals will help to refine our current models of the receptor thus allowing for further advancement in our understanding the nicotinic system and the function of receptors in general.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32NS043063-01
Application #
6446379
Study Section
Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
Program Officer
Nichols, Paul L
Project Start
2002-07-01
Project End
2005-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
1
Fiscal Year
2002
Total Cost
$38,320
Indirect Cost
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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