An α4β4 Nicotinic Receptor Subtype Is Present in Chick Retina: Identification, Characterization and Pharmacological Comparison with the Transfected α4β4 and α6β4 Subtypes
- Benedetta Barabino1,2,
- Silvia Vailati3,
- Milena Moretti3,
- J. Michael McIntosh4,
- Renato Longhi5,
- Francesco Clementi3 and
- Cecilia Gotti3
- 1Department of Experimental Medicine and Pathology, La Sapienza University, Rome (B.B.); 2Department of Biochemistry, Science II, University of Geneva, Geneva, Switzerland (B.B.); 3CNR Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milan, Milan, Italy (S.V., M.M., F.C., C.G.); 4Department of Biology, University of Utah, Salt Lake City, Utah (J.M.M.);5and CNR Center for Hormone Chemistry, Milan, Italy (R.L.)
Abstract
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 β4 subunit associated with different α subunits. Using a sequential immunodepletion procedure with anti-α6, anti-β3, anti-β2, and anti-β4 antibodies, we purified an α4β4 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 α4 and β4 subunits. This receptor binds a number of agonists and the antagonist dihydro-β-erythroidine with nanomolar affinity, whereas it has micromolar affinity for the α-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 absoluteKi values for several nicotinic drugs. Finally, because chick retina expresses an α6β4-containing subtype with a high affinity for the α-conotoxin MII, we used native and transfected α4β4 and α6β4 subtypes to investigate the relative contributions of the α and β subunits to this binding, and found that the α6 subunit determines the high affinity for this toxin.
Footnotes
-
Send reprint requests to: Dr. Cecilia Gotti, CNR Cellular and Molecular Pharmacology Center, Via Vanvitelli 32, 20129 Milano, Italy. E-mail: c.gotti{at}csfic.mi.cnr.it
-
This work was supported in part by grants to F.C. from the Italian Ministry of University and Scientific and Technological Research (MM05152538) and from the European “Training and Mobility of Researchers” Program (contract ERB4061PL97-0790).
-
B.B. and S.V. contributed equally to this work.
- Abbreviations:
- ACh
- acetylcholine
- CNS
- central nervous system
- nAChR
- neuronal nicotinic acetylcholine receptor
- COOH
- subunit COOH peptide
- CYT
- subunit cytoplasmic peptide
- Ab
- polyclonal antibody
- PMSF
- phenylmethylsulfonyl fluoride
- Epi
- epibatidine
- MII
- α-conotoxin MII
- αBgtx
- α-bungarotoxin
- Carb
- carbamylcholine
- Cyt
- cytisine
- DMPP
- 1,1-dimethyl-4-phenylpiperazinium
- Nic
- nicotine
- MLA
- methyllycaconitine
- DHβE
- dihydro-β-erythroidine
- d-TC
- d-tubocurarine
- Hex
- hexamethonium
- Dec
- decamethonium
- CV
- coefficient of variation
-
- Received December 4, 2000.
- Accepted February 14, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
