Antibodies raised against the eel electric organ nAChR were first tested against neuronal nAChRs by Patrick and Stallcup (1977). This antibody caused a functional block of agonist-induced Na+ influx in PC12 cells, but did not recognize the a-BnTx binding site and confirmed pharmacological evidence that the functional neuronal nAChR and the a-BnTx sites are distinct. More importantly, the study indicated that neuronal nAChRs have antigenic similarities to muscle nAChRs.
This similarity was further examined by Jacob et al. (1984); using a mAb to the "main immunogenic region" of an AChR purified from eel electric organ (mAb 35), they tested for cross reactivity with the neuronal nAChR in chick CG neurons (dissociated and cultured) by immunolabeling with HRP-conjugated mAb. The mAb 35 labeled synaptic regions and differed from binding of HRP-conjugated a-BnTx. Subsequently, it was found that the antigen recognized by mAb 35 is found in sympathetic ganglia as well as in CG neurons. In CG, the target protein sediments as a 10S species, consistent with a pentameric receptor (Smith et al., 1985). In fact, the mAb 35 site is the same site targeted by neuronal bungarotoxin (Halvorsen and Berg, 1987). Confirmation that mAbs to nAChRs could recognize extrasynaptic, as well as synaptic, regions in ganglion neurons also came from Sargent and Pang (1989).
Using the specific mAbs developed by Lindstrom and colleagues, Berg and colleagues have begun to elucidate the subunits contributing to native neuronal nAChRs in the chick CG. Beginning with immunopurification of nAChRs from CG and using mAb 35 sepharose, adsorbed proteins were then radiolabeled for autora-diography and immunoprecipitated to determine protein identity and homology with other known nAChR subunits (Halvorsen and Berg, 1990). Three proteins were obtained: a3 plus two unknowns. Following this, Conroy et al. (1992) identified one of the proteins found in chick CG as the a5 subunit by in vitro translation of a5 gene and immunoprecipitation of the synthesized protein to identify a5-specific mAbs. Native proteins, immunopurified on mAb 35 Actigel, could then be immu-noprecipitated to determine whether other subunits co-assemble with a5 in native receptors. Sequential immunoprecipitation and immunodepletion experiments were used to confirm co-assembly of different a subunits: immunodepletion of a3 or ß4 causes less a5 to be recovered.
Subunit composition in the CG was further elucidated by Vernallis et al. (1993). Following immunopurification of nAChRs from CG with mAb 35, immunoprecipitation with specific mAbs identified a3, a5, and ß4; mAb 35 binding was measured after immunoprecipitation with specific mAbs to determine the proportions of subunit-containing receptors in CG. Immunodepletion on specific mAb Actigel followed by immunoblot analysis with other subunit mAbs was used to determine which subunits co-assemble. The study indicated that synaptic receptors contain at least three types of subunits (a3, a5, 04), while extrasynaptic receptors contain a7, but lack a3, a5, and 04 type subunits. These findings were extended by the discovery that CG neurons also contain 02 subunits (Conroy and Berg, 1995). Using a similar approach, 02 was immunopurified and, using mAb immunohistochemistry, demonstrated that 02 mAb labels most neuronal soma, comparable to mAb 35 labeling. Immunoblot and immunodepletion analysis showed that 02-containing receptors are a subpopulation of a304-containing receptors. It was concluded that all synaptic neuronal nAChRs that have a3 also have 04, and vice versa; most a5 is associated with a3 and 04, and 20% of these receptors contain 02. Thus, chick CG neurons express at least 3, and in some cases, 4 different synaptic subunits. Thus, in the chick CG, a3, a5, a7, 02, and 04 subunit proteins have been detected, which corresponds well with the detection of subunit mRNA.
Laser scanning confocal microscopy of mAb 35 and Cy3-conjugated a-BnTx in chick CG revealed that both receptor types were found in clusters widely distributed in embryonic and adult CG neurons in whole-mounted ganglia, while mAb 35 receptors alone showed a punctate distribution (Wilson-Horch and Sargent 1995). There was no evidence for colocalization of a-BnTx with synaptic vesicle proteins, although the clusters were close to synaptic sites, indicating that a-BnTx sites are perisynaptic. Clusters of mAb 35 binding were not synaptic, but punctate areas of mAb 35 binding showed overlap with the synaptic vesicle protein, while nonsynaptic mAb 35 protein overlapped with a-BnTx protein.
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