Nicotinic Cholinergic Receptors in the Rat Retina: Simple and Mixed Heteromeric Subtypes

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Abstract
Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in the rat retina to determine the heteromeric subtypes. We detected seven nicotinic receptor subunit mRNA transcripts, α2–α4, α6, and β2–β4, with RNase protection assays. The density of heteromeric nAChR binding sites is ∼3 times higher in the retina than in the cerebral cortex. Moreover, the density of the sites in the retina measured with [3H]epibatidine ([3H]EB) is ∼30% higher than with 125I-3-(2(S)-azetidinylmethoxy)pyridine (A-85380) and more than twice that measured with [3H]cytisine or [3H](-)nicotine. These data suggest that the retina expresses multiple subtypes of nAChRs, including a large fraction of receptors containing the β2 subunit and a smaller fraction containing the β4 subunit. Consistent with this, in binding competition studies, nicotinic ligands fit a model for two affinity classes of binding sites, with the higher affinity sites representing 70 to 80% of the nAChRs in the retina. To determine the specific subtypes of nAChRs in the rat retina, we used subunit-specific antibodies in immunoprecipitation assays. Immunoprecipitation of [3H]EB-labeled nAChRs with antibodies specific to the β2 and β4 subunits indicated that ∼80% of the receptors contained β2 subunits and ∼25% contained β4 receptors, consistent with the binding pharmacology results. Sequential immunoprecipitation assays indicated that the rat retina contains multiple subtypes of nAChRs. The majority of the receptors measured seemed to be simple heteromeric subtypes, composed of a single type of α and a single type of β subunit; but a significant fraction are mixed heteromeric subtypes, composed of two or more α and/or β subunits.