Functional α6-Containing Nicotinic Receptors Are Present in Chick Retina
- Silvia Vailati1,
- Wolfgang Hanke2,
- Andreea Bejan1,
- Benedetta Barabino3,
- Renato Longhi4,
- Barbara Balestra1,
- Milena Moretti1,
- Francesco Clementi1 and
- Cecilia Gotti1
- 1Consiglio Nazionale delle Ricerche (CNR) Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milan, Milan, Italy (S.V., A.B., Ba.B., M.M., F.C., C.G.); 2Institute of Zoophysiology, Hohenheim University, Stuttgart, Germany (W.H.);3Department of Experimental Medicine and Pathology, Università di Roma “La Sapienza,” Rome, Italy (Be.B.); and 4CNR Center of Hormone Chemistry, Milan, Italy (R.L.)
Abstract
Despite the fact that the neuronal chick α6 subunit was first cloned several years ago and recently has been shown to form acetylcholine (ACh)-activated channels in heterologous systems, no information is yet available concerning the structure and function of the α6-containing nicotinic receptors in neuronal tissues. Using subunit-specific antibodies directed against two different epitopes of the chick α6 subunit, we performed immunoprecipitation experiments on immunopurified α6-containing receptors radiolabeled with the nicotinic agonist [3H]epibatidine (Epi): almost all of the α6 receptors contained the β4 subunit, 51% the β3 subunit, 42% the α3 subunit, and 7.5% the β2 subunit. Western blot analyses of the purified receptors confirmed the presence of the α3, β3, β2, and β4 subunits, and the absence of the α4, α5, and α7 subunits. The α6-containing receptors bind [3H]Epi (Kd = 35 pM) and a number of other nicotinic agonists with very high affinity, the rank order being Epi ≫ cytisine > nicotine > 1,1-dimethyl-4-phenylpiperazinium > acetylcholine > carbamylcholine. The α6 receptors also have a distinct antagonist pharmacological profile with a rank order of potency of α-conotoxin MII > methyllycaconitine > dihydro-β-erythroydine > MG624 > d-tubocurarine > decamethonium > hexamethonium. When reconstituted in lipid bilayers, the α6-containing receptors form functional cationic channels with a main conductance state of 48 pS. These channels are activated by nicotinic agonists in a dose-dependent manner, and blocked by the nicotinic antagonist d-tubocurarine.
Footnotes
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Send reprint requests to: Dr. Cecilia Gotti, Consiglio Nazionale della Ricerche, Cellular Molecular Pharmacology Center, Via Vanvitelli 32, 20129 Milano, Italy. E-mail: Gotti{at}Farma4.csfic.mi.cnr.it
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This work was supported in part by grants from Fabriques de Tabac Réunies, Neuchâtel, Switzerland, the Italian Ministry of University and Scientific and Technological Research, the European Program “Training and Mobility of Researchers,” Contract ERB4061PL97–0790 and the Telethon Grant 1047 to C.G.
- Abbreviations:
- nAChR
- neuronal nicotinic acetylcholine receptor
- αBgtx
- α-bungarotoxin
- ACh
- acetylcholine, Carb, carbamylcholine
- COOH
- subunit COOH peptide
- Cyt
- cytisine
- CYT
- subunit cytoplasmic peptide
- Epi
- epibatidine
- DMPP
- 1,1-dimethyl-4-phenylpiperazinium
- Nic
- nicotine
- MLA
- methyllycaconitine
- DHβE
- dihydro-β-erythroidine
- MII
- α-conotoxin MII
- d-TC
- d-tubocurarine
- COL
- chick optic lobe
- Abs
- polyclonal antibodies
- mAbs
- monoclonal antibodies
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- Received January 11, 1999.
- Accepted April 15, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
