Alpha subunit composition of nicotinic acetylcholine receptors in the rat autonomic ganglia neurons as determined with subunit-specific anti-α(181–192) peptide antibodies

Abstract

The subunit composition of nicotinic acetylcholine receptors of rat autonomic ganglia neurons was studied by means of antibodies, which differentiated between different α subunits and specifically blocked acetylcholine-induced membrane currents. Polyclonal rabbit antibodies and mouse monoclonal antibodies were raised against synthetic peptides matching in sequence the α(181–192) region of α3, α4, α5, and α7 subunits of rat neuronal nicotinic acetylcholine receptors. The antibodies discriminated among α3, α4, α5, and α7 peptides in enzyme-linked immunosorbent assay and bound to native acetylcholine receptors expressed in PC-12 cells. By means of immunoperoxidase staining of cultured rat autonomic neurons followed by transmission, dark-field and phase-contrast microscopy, it was found that all cells of the superior cervical ganglia expressed the α3, α5, and α7 nicotinic acetylcholine receptors, whereas approximately half of the cells were clearly α4-positive. In contrast, only about one-third of the intracardiac neurons were α3-positive, about 50% were α4-positive, one-seventh were α5-positive, and one-fifth were α7-positive. All antibodies tested blocked acetylcholine-induced currents in the neurons of the superior cervical ganglia as was demonstrated by whole-cell patch-clamp studies. Although each antibody could block up to 80% of the current, the degree of inhibition varied considerably from cell to cell. It is concluded that α3, α5, and α7 subunits are expressed in all neurons of the superior cervical ganglion and in some intracardiac neurons, whereas α4 subunits are expressed in some but not all neurons of both tissues. The neurons of the superior cervical ganglion express heterogeneous acetylcholine receptors and differ in relative amounts of acetylcholine receptor subtypes expressed.