alpha 7 and alpha 8 Nicotinic Receptor Subtypes Immunopurified from Chick Retina have Different Immunological, Pharmacological and Functional Properties

• Published in: The European journal of neuroscience 1997-06, Vol.9 (6), p.1201-1211
• Main Authors: Gotti, Cecilia, Moretti, Milena, Maggi, Roberto, Longhi, Renato, Hanke, Wolfang, Klinke, Nicole, Clementi, Francesco
• Format: Article
• Language: English
• ISSN: 0953-816X; 1460-9568
• DOI: 10.1111/j.1460-9568.1997.tb01475.x

Nicotinic receptors are present in the chick retina, but their structure and functional characteristics are still unclear. Using anti- alpha 7 and anti- alpha 8 subunit-specific antibodies, we immunopurified the alpha 7 and alpha 8 subtypes of chick retina neuronal nicotinic receptors. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the two purified subtypes consistently showed a similar peptide composition characterized by the presence of two major peptides of M sub(r) 58 plus or minus 1 and 54 plus or minus 1 kDa, and two minor peptides of 67 and 61 plus or minus 1 kDa. In the alpha 7 subtype, the 58 kDa peptide was recognized by anti- alpha 7 but not by anti- alpha 8 antibodies; in the alpha 8 subtype, the 58 kDa peptide was recognized only by anti- alpha 8 antibodies. The alpha 7 subtype had a single class of [ super(125)]a-bungarotoxin binding sites with a KD value of 1.2 nM, whereas the purified alpha 8 subtype had two classes of binding sites, one with a K sub(D) of 5.5 nM and the other with very high affinity (K sub(D) 52 pM), but present in only 8% of the receptors. Competition binding experiments also showed the presence on the alpha 8 subtype of highand low-affinity classes of binding sites; the affinity for cholinergic drugs of the former was greater than that of the single class present on the alpha 7 subtype. When reconstituted in planar lipid bilayers, both subtypes formed ligand-gated cation channels with major conductance levels of 42 and 52 pS but with different lifetimes; the two channels were activated by agonists and blocked by d-tubocurarine and the glycinergic antagonist strychnine. In line with the binding data, the reconstituted alpha 8 subtype had greater agonist sensitivity than the alpha 7 subtype.