Formation of Oligomers Containing the β3 and β4 Subunits of the Rat Nicotinic Receptor

Abstract

The role of the ␤3 and ␤4 subunits of the nicotinic acetylcholine receptor in brain is still unclear. We investigated nicotinic receptor structure with antibodies directed against unique regions of the ␤3 and ␤4 subunits of the rat nicotinic acetylcholine receptor. Anti-␤4 detected a single band of 66 kDa in most regions of the brain that was strongest in striatum and cerebellum. The 60 kDa ␤3 subunit was detected primarily in striatum and cerebellum, and faintly in hippocampus. Immunoprecipitation experiments established that the two subunits were coassembled in the cerebellum along with the ␤2 subunit. Antibodies against the ␣4, ␤2, ␤3, and ␤4 subunits immunoprecipitated ϳ75% of the bungarotoxin-insensitive nicotinic receptor from cerebellar extracts as determined by nicotine-dependent acetylcholine binding. Transfection of COS cells with cDNAs for these four subunits induced expression of a high affinity nicotinic receptor. Omission of only a single subunit from the transfection affected either the B max or the apparent K D of the receptor. Our data suggest that the ␤3 subunit functions as a structural entity that links a relatively unstable ␣4␤2 heterodimer to a more stable ␣4␤4 heterodimer. The agonistbinding site formed by ␣4␤2 has a much greater affinity than does that formed by ␣4␤4. In this respect, nicotinic receptors that contain the ␤3 subunit are structurally homologous to the muscle nicotinic receptor.Key words: nicotine; acetylcholine; receptor; antibody; ␤3; ␤4; subunit Nicotinic acetylcholine receptors (nAChR) expressed in the CNS are members of a superfamily of ligand-gated ion channels that also include the muscle-type nAChR, GABA A , glycine, and 5-HT 3 receptors (Barnard et al., 1987;Egebjerg et al., 1991;Maricq et al., 1991;Moriyoshi et al., 1991;Monyer et al., 1992). Nine different rat neuronal nAChR subunit cDNAs (␣2, ␣3, ␣4, ␣5, ␣6, ␣7, ␤2, ␤3, and ␤4) have been cloned (for review, see Sargent, 1993). Most of the receptors in the family are thought to contain two or more different subunits, but the precise combinations of these subunits that exist in vivo are largely unknown. Previous work has indicated that the major high affinity nicotinic receptor in rat brain is composed of ␣4 and ␤2 subunits (Nakayama et al., 1991;Flores et al., 1992;Marks et al., 1992). More recent evidence indicates that deletion of the ␤2 subunit gene results in the loss of high affinity nicotine-binding sites from mouse brain (Picciotto et al., 1995). Nicotinic receptors have also been reconstituted in Xenopus oocytes. Thus, the ␤2 and ␤4 subunits can combine with various ␣ subunits to form functional receptors, permitting the formation of many types of receptor with unique pharmacological characteristics (Luetje and Patrick, 1991). Much less is known about the ␤3 subunit. It does not express any channel activity in oocytes in combination with any other single subunit , nor has it been demonstrated at the protein level in the CNS. Both the ␤3 and ␤4 subunits seem, by in situ hybridization, to have a more restric...

Nicotinic Cholinergic Receptors in the Rat Cerebellum: Multiple Heteromeric Subtypes

“…Although a previous study reported the presence of nAChRs containing ␤3 subunits in association with ␤4 subunits in the rat cerebellum (Forsayeth and Kobrin, 1997), we did not find evidence for the ␤3 subunit mRNA or protein in our studies. It is possible that the ␤3 subunit is expressed but at levels below our detection limits.…”

Cloning and Functional Characterisation of Two Novel Nicotinic Acetylcholine Receptor α Subunits from the Insect Pest Myzus persicae

“…At the vertebrate neuromuscular junction, nAChRs have also been shown to have a functional multisubunit composition with a generic stoichiometry of 2a1;/31;y/E;ö. Vertebrate neuronal nAChRs constitute a more diverse family of receptors. Although the basic pentameric structure is maintained (Anand et al, 1991), these probably assemble according to a 2a13/ 3 stoichiometry with possibly more than one aor a-subunit subtype within individual receptors (Galzi and Changeux, 1995;Forsayeth and Kobrin, 1997). However, three neuronal a subunits (a7-a9) have also been shown to form functional homo-oligomeric channels in a range of heterologous expression systems (Elgoyhen et al, 1994;Gerzanich Ct al., 1994).…”

Functional Properties of the Neuronal Nicotinic Acetylcholine Receptor β3 Promoter in the Developing Central Nervous System

“…Forsayeth and Kobrin (16) have shown that the ␤3 subunit co-assembles in vivo with the ␣4, ␤2, and ␤4 subunits to form a functional nicotinic receptor endowed with distinctive properties. We have isolated a short 5Ј-sequence of the ␤3 gene containing promoter elements that are sufficient to target reporter gene expression to those retinal neurons that normally express ␤3 in vivo (11,15).…”