RESUMEN
The monoclonal antibody M35, one of the first monoclonal antibodies successfully raised against muscarinic acetylcholine receptors, has been widely used to study the distribution of this protein in a variety of tissues and cell types of different species. It is not fully known, however, to which muscarinic acetylcholine receptor subtypes M35 binds. Knowledge of subtype-selectivity of M35 is a necessary step towards a functional interpretation of the obtained immunocytochemical data. The aim of the present study was to determine the subtype-selectivity of M35 employing transfected CHO-K1 cells stably expressing human m1-m5 muscarinic acetylcholine receptors separately, and to study M35 immunoreactivity in areas of rat central and peripheral tissues known to be specifically enriched in a single muscarinic acetylcholine receptor subtype. The results show that (a) all five transfected cell lines were immunopositive for M35, (b) nontransfected control cells were immunonegative, (c) the number of mAChRs expressed per cell correlated positively with the intensity of M35 immunoreactivity, and (d) cell types in aldehyde-fixed rat tissue enriched in a single m1-m4 subtypes revealed clear M35 immunoreactivity. Taken together, the present results show that M35 does not discriminate between muscarinic acetylcholine receptor subtypes. Evidently, the epitope of M35 on the receptor-protein is preserved on all muscarinic acetylcholine receptor subtypes. The epitope for M35 must, therefore, be localized on a homologous part of each subtype.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptores Muscarínicos/inmunología , Animales , Células CHO , Cricetinae , Humanos , Ratas , Ratas Wistar , Receptores Muscarínicos/clasificaciónRESUMEN
Toxins from the venom of the African green mamba, Dendroaspis angusticeps, fulfill a major need for selective ligands for some of the five genetically defined subtypes of muscarinic acetylcholine receptors (m1-m5). Two toxins have been found that are highly selective antagonists for m1 and m4 receptors (m1-toxin and m4-toxin, respectively). Two other toxins (MT1 and MT2) bind with high affinity to both m1 and m4 receptors, and are agonists. Components of the venom also modify the binding of radiolabeled antagonists to m2 receptors, but an m2-selective toxin has not yet been isolated, m1-Toxin can bind to m1 receptors at the same time as typical competitive antagonists, suggesting that this toxin binds to the N-terminal and outer loops of m1 receptor molecules, rather than within the receptor pocket where typical agonists and antagonists bind. The binding of toxins to the outer parts of receptor molecules probably accounts for their much higher specificity for individual receptor subtypes than is seen with smaller ligands. Toxins are useful for identifying, counting, localizing, activating and blocking m1 and m4 receptors with high specificity.