Vol. 59, Issue 6, 1410-1417, June 2001

An 44 Nicotinic Receptor Subtype Is Present in Chick Retina: Identification, Characterization and Pharmacological Comparison with the Transfected 44 and 64 Subtypes

Benedetta Barabino, Silvia Vailati, Milena Moretti, J. Michael McIntosh, Renato Longhi, Francesco Clementi, and Cecilia Gotti

Department of Experimental Medicine and Pathology, La Sapienza University, Rome (B.B.); Department of Biochemistry, Science II, University of Geneva, Geneva, Switzerland (B.B.); CNR Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milan, Milan, Italy (S.V., M.M., F.C., C.G.); Department of Biology, University of Utah, Salt Lake City, Utah (J.M.M.); and CNR Center for Hormone Chemistry, Milan, Italy (R.L.)

Retina from 1-day-old chicks is a valuable tissue model for studying neuronal nicotinic receptors because it expresses a large number of the developmentally regulated high affinity [³H]epibatidine labeled nicotinic receptors. Most of these receptors contain the 4 subunit associated with different  subunits. Using a sequential immunodepletion procedure with anti-6, anti-3, anti-2, and anti-4 antibodies, we purified an 44 nicotinic receptor subtype that accounts for approximately 20 to 25% of the high affinity [³H]epibatidine labeled receptors present in retina at that developmental time. Immunoprecipitation and Western blotting experiments confirmed that the purified subtype contains only the 4 and 4 subunits. This receptor binds a number of agonists and the antagonist dihydro--erythroidine with nanomolar affinity, whereas it has micromolar affinity for the -conotoxin MII and methyllycaconitine toxins and other nicotinic antagonists. Comparison of the pharmacological profile of this purified native subtype with that of the same subtype transiently expressed in human BOSC23 cells showed that they have very similar rank orders and absolute Ki values for several nicotinic drugs. Finally, because chick retina expresses an 64-containing subtype with a high affinity for the -conotoxin MII, we used native and transfected 44 and 64 subtypes to investigate the relative contributions of the  and  subunits to this binding, and found that the 6 subunit determines the high affinity for this toxin.