Vol. 58, Issue 2, 300-311, August 2000

Chick Optic Lobe Contains a Developmentally Regulated 252 Nicotinic Receptor Subtype¹

Barbara Balestra, Silvia Vailati, Milena Moretti, Wolfang Hanke, Francesco Clementi, and Cecilia Gotti

Consiglio Nazionale delle Ricerche Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milan, Milan, Italy (B.B., S.V., M.M., F.C., C.G.); and Institut für Zoophysiologie, Universität Hohenheim, Stuttgart, Germany (W.H.)

The most widely expressed neuronal nicotinic acetylcholine receptor subtype in chick brain is that containing the 4 and 2 subunits. However, immunoprecipitation and localization studies have shown that some brain areas also contain the 2 and/or 5 subunits, whose role in the definition of receptor properties is still intriguing. Using subunit-specific polyclonal antibodies, we found that the optic lobe is the chick central nervous system region that expresses the highest level of 2-containing receptors. Immunoprecipitation studies of these immunopurified 2-containing receptors labeled with the nicotinic agonist [³H]epibatidine showed that almost all of them contained the 2 subunit and that more than 66% contained the 5 subunit. Western blot analyses of the purified receptors confirmed the presence of the 2, 5, and 2 subunits and the absence of the 3, 4, 6, 7, 8, 3, and 4 subunits. The 2-containing receptors are developmentally regulated: their expression increases 25 times from embryonic day 7 to posthatching day 1 in the optic lobe, compared with an increase of only 5-fold in the forebrain. The 2-containing optic lobe receptors bind [³H]epibatidine (K_d = 29 pM) and a number of other nicotinic agonists with very high affinity and have a pharmacological profile very similar to that of the 42 subtype. They form functional cationic channels when reconstituted in lipid bilayers, with pharmacological and biophysical properties different from those of the 42 subtype. These channels are activated by nicotinic agonists in a dose-dependent manner and are blocked by the nicotinic antagonist d-tubocurarine.