An alpha 4 beta 4 nicotinic receptor subtype is present in chick retina: Identification, characterization and pharmacological comparison with the transfected alpha 4 beta 4 and alpha 6 beta 4 subtypes

Retina from 1-day-old chicks is a valuable tissue model for studying neuron al nicotinic receptors because it expresses a large number of the developme ntally regulated high affinity [H-3]epibatidine labeled nicotinic receptors . Most of these receptors contain the beta4 subunit associated with differe nt alpha subunits. Using a sequential immunodepletion procedure with anti-a lpha6, anti-beta3, anti-beta2, and anti-beta4 antibodies, we purified an al pha4 beta4 nicotinic receptor subtype that accounts for approximately 20 to 25% of the high affinity [H-3]epibatidine labeled receptors present in ret ina at that developmental time. Immunoprecipitation and Western blotting ex periments confirmed that the purified subtype contains only the alpha4 and beta4 subunits. This receptor binds a number of agonists and the antagonist dihydro-beta -erythroidine with nanomolar affinity, whereas it has micromo lar affinity for the alpha -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 expr essed 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 alpha6 beta4-containing subtype with a high affinity fo r the alpha -conotoxin MII, we used native and transfected alpha4 beta4 and alpha6 beta4 subtypes to investigate the relative contributions of the alp ha and beta subunits to this binding, and found that the alpha6 subunit det ermines the high affinity for this toxin.