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(Accepted August 20, 2002)
Comparative structure-function studies have been carried out for a-conotoxin GI acting on nicotinic acetylcholine receptors (AChR) from mammalian muscles and from the electric organ of the Torpedo californica ray and for a-conotoxin ImI, which targets the neuronal a7 AChR. A series of analogs has been prepared for this purpose: chemically modified derivatives, including a covalently linked dimer of GI, as well as analogs wherein one or several amino acid residues have been changed using solid-phase peptide synthesis. The activity of all compounds was assessed in competition with radioiodinated and/or tritiated a-conotoxin GI for binding to the membranebound AChR of Torpedo californica. Binding of radioiodinated a-conotoxin GI dimer was also monitored directly, revealing the largest, as compared to all other analogues, difference in the affinity between the two binding sites in the receptor (KD ,11 and 1200 nM). Comparison of binding data with the results of CD measurements point to important role of the spatial organization of the a-conotoxin second loop in manifestation of their "muscle" or "neuronal" specificity.
KEY WORDS: a-Conotoxins; analogues; nicotinic acetylcholine receptor; radioligand assay; CD spectroscopy.
INTRODUCTION
a-Conotoxins from the Conus marine snails interact with the binding sites for agonists/competitive antagonists of various AChRs. A characteristic feature of a-conotoxins is the selectivity toward distinct, neuronal or muscle-type AChRs, as well as species specificity (see reviews 1,2). As compared to a-neurotoxins from snake venoms, a-conotoxins discriminate much better two binding sites on the muscle-type AChRs (3-5). a-Conotoxins targeting the neuronal AChRs distinguish their various subtypes (6-10), but the stoichiometry of such an interaction is less clear. Because a-conotoxins are relatively short peptides, their unsurpassed selectivity makes them a unique instrument for studies of AChRs. Spatial structures of many a-conotoxins have been determined by NMR and X-ray analyses (see, e.g., 11-13). Additional advantages of a-conotoxins are a relatively simple chemical synthesis and the possibility to prepare diverse analogues (5,14-16).
We have earlier prepared a series of photoactivatable analogues of a-conotoxins GI and MI to probe the topography of toxin-binding sites in the Torpedo AChR (17,18). In the present work, we tested biological activity of a series of new analogs of "muscle-acting" aconotoxin GI and "neuronal-acting" a-conotoxin ImI (which targets the neuronal homooligomeric a7 AChR) to shed light on the...