Alpha7 and alpha8 nicotinic receptor subtypes immunopurified from chick retina have different immunological, pharmacological and functional properties.
Nicotinic receptors are present in the chick retina, but their structure and functional characteristics are still unclear. Using anti-alpha7 and anti-alpha8 subunit-specific antibodies, we immunopurified the alpha7 and alpha8 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(r) 58 +/- 1 and 54 +/- 1 kDa, and two minor peptides of 67 and 61 +/- 1 kDa. In the alpha7 subtype, the 58 kDa peptide was recognized by anti-alpha7 but not by anti-alpha8 antibodies; in the alpha8 subtype, the 58 kDa peptide was recognized only by anti-alpha8 antibodies. The alpha7 subtype had a single class of [125I]alpha-bungarotoxin binding sites with a K(D) value of 1.2 nM, whereas the purified alpha8 subtype had two classes of binding sites, one with a K(D) of 5.5 nM and the other with very high affinity (KD 52 pM), but present in only 8% of the receptors. Competition binding experiments also showed the presence on the alpha8 subtype of high- and 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 alpha7 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 alpha8 subtype had greater agonist sensitivity than the alpha7 subtype.
Gotti
C
C
CNR Center of Cellular and Molecular Pharmacology, Department of Medical Pharmacology, University of Milan, Italy.
Moretti
M
M
Maggi
R
R
Longhi
R
R
Hanke
W
W
Klinke
N
N
Clementi
F
F
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002038:Bungarotoxins / Q000378:metabolism; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D008024:Ligands; D008051:Lipid Bilayers; D018722:Nicotinic Agonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000302:isolation & purification; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D012160:Retina / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D014101:Torpedo / Q000502:physiology*
D002038:Bungarotoxins; D008024:Ligands; D008051:Lipid Bilayers; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D010455:Peptides; D011978:Receptors, Nicotinic