Alpha subunit composition of nicotinic acetylcholine receptors in the rat autonomic ganglia neurons as determined with subunit-specific anti-α(181–192) peptide antibodies
Section snippets
Materials
Peroxidase-conjugated goat anti-mouse and anti-rabbit IgG, peroxidase–antiperoxidase complex, collagenase from Clostridium histolyticum, type IA, Freund's adjuvants and culture media were from Sigma, U.S.A.; AH-Sepharose 4B was from Pharmacia Fine Chemicals, Sweden; fetal calf serum was either from Seromed, Germany, or from Sigma, U.S.A.; fluorescein isothiocyanate (FITC)-labelled goat anti-mouse or goat anti-rabbit IgG were from Coulter Clone, France. BALB/c mice were bred in the Palladin
Antibodies to α(181–192) peptides discriminate among the peptides of different α subunits
The sera of immunized rabbits obtained after two rounds of immunization strongly preferred to bind α(181–192) peptides used for immunization and poorly bound corresponding peptides of other subunits, as studied by the enzyme-linked immunosorbent assay. The potential cross-reactivity was minimized to negligible values following the antibody affinity purification. Monoclonal antibody (MAb) 1D6-3-8 (IgG2a), obtained against α3 peptide, did not bind other peptides (Table 2). MAb 5C7 (IgM) obtained
Characterization of the antibodies used
Affinity purified α(181–192)-specific antibodies discriminated among the peptides of different α-subunits in enzyme-linked immunosorbent assay. Flow cytometry and immunocytochemical experiments performed with PC-12 cells and cultured autonomic ganglia neurons revealed that the antibodies bound native AChRs. Relative numbers of PC-12 cells, SCG or ICG cells stained with α(181–192)-specific antibodies were different for different antibodies (Fig. 2). Therefore, the antibodies discriminated not
Conclusions
(1) Antibodies directed against (181–192) fragments of α3, α4, α5 and α7 subunits of neuronal AChR bind native AChRs and block ACh-induced membrane currents in autonomic ganglia. (2) Single SCG neurons expresses heteromeric AChRs; the neurons differ in relative amounts of AChR subtypes expressed. (3) The neurons of SCG and ICG differ markedly in the α subunit composition of their AChRs.
Acknowledgements
The work was supported with PECO grant ERBCIPDCT 940244 and with INTAS-Ukraine grant 95-0056.
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