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Although 10 genes have been cloned encoding putative subunits of neuronal nicotinic acetylcholine receptors, little is known about the variety or subunit composition of such receptors expressed by individual neurons. Chick ciliary ganglion neurons express five of the known genes and assemble a class of synaptic-type receptors collectively containing gene products from three of them: alpha 3, beta 4, and alpha 5. Using subunit-specific monoclonal antibodies, we show here that all of the synaptic-type acetylcholine receptors having alpha 3 also have beta 4 subunits and vice versa. In addition, most, if not all, of the alpha 5 gene product present in fully assembled receptors is associated with both alpha 3 and beta 4 subunits. Although the receptors may be homogeneous in these respects, only about 20% of them also contain the fourth gene product, beta 2, newly identified in the ganglion; essentially all of the neurons express the beta 2 gene. No beta 2 subunits are found coassembled with the fifth acetylcholine receptor gene product expressed by the neurons, alpha 7, which has been shown previously to comprise a class of abundant, nonsynaptic receptors on the cells. The identification of three acetylcholine receptor subtypes distinguished by subunit composition on the same neurons provokes questions about their individual physiological roles.

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Nicotinic receptors are cation-ion selective ligand-gated ion channels that are expressed throughout the nervous system. Most have significant calcium permeabilities, enabling them to regulate calcium-dependent events. One of the most abundant is a species composed of the ␣7 gene product and having a relative calcium permeability equivalent to that of NMDA receptors. The ␣7-containing receptors can be found presynaptically where they modulate transmitter release, and postsynaptically where they generate excitatory responses. They can also be found in perisynaptic locations where they modulate other inputs to the neuron and can activate a variety of downstream signaling pathways. The effects the receptors produce depend critically on the sites at which they are clustered. Instructive preparations for examining ␣7-containing receptors are the rat hippocampus, where they are thought to play a modulatory role, and the chick ciliary ganglion, where they participate in throughput transmission as well as regulatory signaling. Relatively high levels of ␣7-containing receptors are found in the two preparations, and the receptors display a variety of synaptic options and functions in the two cases. Progress is starting to be made in understanding the mechanisms responsible for localizing the receptors at specific sites and in identifying components tethered in the vicinity of the receptors that may facilitate signal transduction and downstream signaling.

Protein scaffolds are essential for specific and efficient downstream signaling at synapses. Though nicotinic receptors are widely expressed in the nervous system and influence numerous cellular events due in part to their calcium permeability, no scaffolds have yet been identified for the receptors in neurons. Here we show that specific members of the PSD-95 family of PDZ-containing proteins are associated with specific nicotinic receptor subtypes. At postsynaptic sites, the PDZ scaffolds are essential for maturation of functional nicotinic synapses on neurons. They also help mediate downstream signaling as exemplified by activation of transcription factors. By tethering components to postsynaptic nicotinic receptors, PDZ scaffolds can organize synaptic structure and determine which calcium-dependent processes will be subject to nicotinic modulation.

A recent report described the isolation of cDNA clones encoding alpha 7 and alpha 8 subunits of alpha-bungarotoxin-sensitive nicotinic ACh receptors (alpha BgtAChRs) from chick brain and demonstrated that they were related to, but distinct from, the alpha subunits of nicotinic ACh receptors (nAChRs) from muscles and neurons. Monoclonal antibodies against the two alpha BgtAChR subunits were used to demonstrate that at least two subtypes are present in embryonic day 18 chicken brain. The predominant brain subtype contains alpha 7 subunits, while a minor subtype contains both alpha 7 and alpha 8 subunits. Both subtypes may also contain other subunits. Here we report the results of immune precipitation studies and immunohistochemical studies of alpha BgtAChRs in the chick retina. In addition to the two subtypes found in brain, a new alpha BgtAChR subtype that contains alpha 8 subunits, but not alpha 7 subunits, was identified and was found to be the major subtype in chick retina. This subtype has a lower affinity for alpha-bungarotoxin (alpha Bgt) than does the subtype containing only alpha 7 subunits. Small amounts of this alpha 8 subtype were also detected in brain by labeling with higher concentrations of 125I-alpha Bgt than had been used previously. The subtype containing only alpha 7 subunits comprised 14% of the alpha BgtAChRs in hatchling chick retina. The subtype containing alpha 8 subunits (but no alpha 7 subunits) accounted for 69%, and the alpha 7 alpha 8 subtype accounted for 17%. Amacrine, bipolar, and ganglion cells displayed alpha 8 subunit immunoreactivity, and a complex pattern of labeling was evident in both the inner and outer plexiform layers. In contrast, only amacrine and ganglion cells exhibited alpha 7 subunit immunoreactivity, and the pattern of alpha 7 subunit labeling in the inner plexiform layer differed from that of alpha 8 subunit labeling. These disparities suggest that the alpha BgtAChR subunits are differentially expressed by different populations of retinal neurons. In addition, the distribution of alpha BgtAChR subunit immunoreactivity was found to differ from that of alpha-Bgt-insensitive nAChR subunits.

A major class of nicotinic receptors in the nervous system is one that binds ␣-bungarotoxin and contains the ␣7 gene product. PC12 cells, frequently used to study nicotinic receptors, express the ␣7 gene and have binding sites for the toxin, but previous attempts to elicit currents from the putative receptors have failed. Using whole-cell patch-clamp recording techniques and rapid application of agonist, we find a rapidly desensitizing acetylcholine-induced current in the cells that can be blocked by ␣-bungarotoxin. The current amplitude varies dramatically among three populations of PC12 cells but correlates well with the number of toxin-binding receptors. In contrast, the current shows no correlation with ␣7 transcript; cells with high levels of ␣7 mRNA can be negative for toxin binding and yet have other functional nicotinic receptors. Northern blot analysis and reverse transcription-PCR reveal no defects in ␣7 RNA from the negative cells, and immunoblot analysis demonstrates that they contain full-length ␣7 protein, although at reduced levels. Affinity purification of toxin-binding receptors from cells expressing them confirms that the receptors contain ␣7 protein. Transfection experiments demonstrate that PC12 cells lacking native toxin-binding receptors are deficient at producing receptors from ␣7 gene constructs, although the same cells can produce receptors from other transfected gene constructs. The results indicate that nicotinic receptors that bind ␣-bungarotoxin and contain ␣7 subunits require additional gene products to facilitate assembly and stabilization of the receptors. PC12 cells offer a model system for identifying those gene products.