Nicotinic signal transduction machinery.

Nicotinic synapses employ acetylcholine to activate ligand-gated ion channels that are cation-selective in vertebrates. Although the resulting nicotinic cholinergic transmission is famously excitatory at the neuromuscular junction, it plays many additional roles in the CNS. Most prevalent is that of modulation, usually involving calcium and signal transduction. Because of this, it is becoming increasingly important not only to understand the mechanisms that guide nicotinic receptors to appropriate locations but also to identify the postsynaptic machinery making possible the requisite signal transduction. Clearly, the kinds of components tethered in the vicinity of the receptor will assume a major role in determining the consequences of receptor activation. One of the most abundant and interesting nicotinic receptors in this respect is the species comprised of the alpha7 gene product (Broide and Leslie, 1999). These alpha7 homopentameric nicotinic acetylcholine receptors (alpha7 nAChRs) have a high relative permeability to calcium, rivaling that of NMDA receptors. But unlike NMDA receptors, alpha7 nAChRs promote calcium influx without requiring a coincident event such as membrane depolarization. As a result, the receptors are well equipped to regulate calcium-dependent events in neurons, particularly when depolarization might be occluded.

Cluster formation of alpha7-containing nicotinic receptors at interneuronal interfaces in cell culture.

Nicotinic receptors containing the alpha7 gene product are among the most abundant in the nervous system. Because of their widespread distribution and high relative permeability to calcium, the receptors regulate a diverse array of cellular events. On chick ciliary neurons the receptors are concentrated on somatic spines folded into discrete mats on the cell body and are overlaid by a large presynaptic calyx. The receptors co-localize with filamentous actin and the actin-associated protein drebrin which are concentrated in the spines. We show here that embryonic ciliary ganglion neurons grown in dissociated cell culture express and concentrate the receptors in large clusters or plaques that form at interneuronal interfaces between small clumps of neurons. The receptors resist detergent extraction even after disruption of the actin cytoskeleton, suggesting the importance of additional molecular mechanisms determining receptor location. The cell adhesion molecules N-CAM and N-cadherin are concentrated at the receptor plaques and may influence plaque stability. Although ciliary neurons do not normally contact each other in vivo, they do so in culture and may mimic interactions normally occurring between calyx and soma in vivo. As a result the cultures may prove useful for identifying components shaping development of postsynaptic specializations on neurons.

The neurofilament infrastructure of a developing presynaptic calyx.

Calyx-type synapses appear to be specifically designed to support fast, reliable, high-frequency excitatory transmission. In the chick ciliary ganglion, calyx terminals from preganglionic neurons in the midbrain form early in development on ciliary neurons. We find that labeling the calyx membranes with a lipophilic dye delivered by diffusion down the preganglionic nerve reveals a large membrane structure engulfing the postsynaptic cell by the end of embryogenesis. In contrast, labeling the calyces with a water-soluble dye by diffusion through the preganglionic nerve suggests large discontinuities in the calyx. A similar pattern of discontinuities is seen when presynaptic neurofilaments are labeled with antibodies selective for highly phosphorylated neurofilaments. The neurofilament infrastructure of the calyx first appears as a single thick bundle, which subsequently bifurcates during development and eventually generates a fine meshwork of filaments subdivided by several large neurofilament bundles encircling the postsynaptic cell body. The large bundles probably produce protruding ridges in the otherwise thin calyx cup, accounting for the disparity in staining patterns observed with membrane and cytosolic dyes. The postsynaptic membrane also undergoes restructuring during development with the appearance of large folded mats of somatic spines heavily invested with nicotinic receptors. The large presynaptic neurofilament bundles do not overlap the postsynaptic receptor clusters but do codistribute with large tracks of presynaptic microtubules. The neurofilament bundles may act as girders to provide structural support while at the same time defining conduits for microtubule-dependent transport of materials and rapid propagation of electrical signals throughout the extended calyx.

Rapsyn variants in ciliary ganglia and their possible effects on clustering of nicotinic receptors.

Nicotinic acetylcholine receptors (nAChRs) containing the alpha7 gene product can influence a range of cellular events in neurons, depending on receptor location. On chick ciliary neurons, the receptors are concentrated on somatic spines, but little is known about mechanisms responsible for sequestering them there. Rapsyn is a 43-kDa protein essential for clustering nicotinic receptors at the vertebrate neuromuscular junction. RT-PCR confirmed previous studies showing that the chick ciliary ganglion expresses rapsyn transcripts, including several splice variants lacking part or all of exon 2. Heterologous expression of rapsyn constructs, together with nicotinic receptor constructs, shows that chicken full-length rapsyn can induce clustering of both muscle and neuronal nicotinic receptors. Splice variants lacking one or both leucine zipper motifs of exon 2 are unable to cluster the receptors, though, like full-length rapsyn, they cluster themselves. Immunological analysis demonstrates the presence of full-length rapsyn in chick muscle extracts but fails to detect either full-length or splice-variant versions of rapsyn at significant levels in ganglion extracts. The results suggest that rapsyn does not cluster alpha7-nAChRs on ciliary neurons in any way similar to that of receptors at the neuromuscular junction where rapsyn and the receptors are present in approximately equimolar amounts.

Organizing effects of rapsyn on neuronal nicotinic acetylcholine receptors.

Targeting receptors to appropriate locations on the cell surface is a critical task for neurons. We have examined the possibility that rapsyn controls the distribution of nicotinic receptors on neurons as it does nicotinic receptors on muscle fibers. Cotransfection of QT6 cells with rapsyn and neuronal nicotinic receptor cDNA constructs produced receptor aggregates or clusters that codistributed in part with rapsyn protein. Though all nicotinic receptor subtypes tested were affected by rapsyn, receptors containing the alpha 7 gene product were among the most responsive. In addition, rapsyn caused a portion of the nicotinic receptors containing alpha 7 subunits to become resistant to solubilization with nonionic detergent and to display a marked increase in metabolic stability. The results are consistent with rapsyn linking the receptors to cytoskeletal elements and suggest that it may play an organizing role determining the fate and location of nicotinic receptors on neurons.

Nicotinic receptor subtypes in the developing chick brain: appearance of a species containing the alpha4, beta2, and alpha5 gene products.

Increasing evidence suggests nicotinic receptors regulate developmental events in the nervous system. We used [3H]epibatidine and 125I-alpha-bungarotoxin, together with subunit-specific monoclonal antibodies, to distinguish and quantify nicotinic receptor subtypes in developing chick brain. The results show that more than three fourths of the epibatidine-binding receptors at both early and late embryonic stages contain alpha4 and beta2 subunits, representing receptors previously distinguished by high affinity nicotine binding. A fraction of these also contain the alpha5 gene product, which is consistent with studies on transfected cells showing that the alpha4, beta2, and alpha5 gene products coassemble to produce epibatidine-binding receptors. A small portion of the receptors contain alpha3 and beta4 subunits, assembled in part with either alpha4 or beta2 subunits. The most abundant nicotinic receptors, however, at both early and late embryonic stages are those having high affinity for alpha-bungarotoxin rather than epibatidine. Most contain alpha7 subunits, whereas about half contain alpha8 subunits as well. The sharpest developmental increase between embryonic days 8 and 17/18 occurs with receptors containing alpha5 subunits, whereas receptors containing alpha3 or beta4 subunits undergo no specific increase. The three major receptor species (containing alpha4 and beta2 but not alpha5 subunits; alpha7 subunits; or alpha7 and alpha8 subunits) each increase approximately 3-fold during the same period. The results indicate greater receptor complexity than appreciated previously; they provide information about the rules governing subunit assembly in neuronal nicotinic receptors and draw attention to the role of alpha5 subunits in late development.

Detection of functional nicotinic receptors blocked by alpha-bungarotoxin on PC12 cells and dependence of their expression on post-translational events.

A major class of nicotinic receptors in the nervous system is one that binds alpha-bungarotoxin and contains the alpha7 gene product. PC12 cells, frequently used to study nicotinic receptors, express the alpha7 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 alpha-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 alpha7 transcript; cells with high levels of alpha7 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 alpha7 RNA from the negative cells, and immunoblot analysis demonstrates that they contain full-length alpha7 protein, although at reduced levels. Affinity purification of toxin-binding receptors from cells expressing them confirms that the receptors contain alpha7 protein. Transfection experiments demonstrate that PC12 cells lacking native toxin-binding receptors are deficient at producing receptors from alpha7 gene constructs, although the same cells can produce receptors from other transfected gene constructs. The results indicate that nicotinic receptors that bind alpha-bungarotoxin and contain alpha7 subunits require additional gene products to facilitate assembly and stabilization of the receptors. PC12 cells offer a model system for identifying those gene products.

Direct recording of nicotinic responses in presynaptic nerve terminals.

Nicotinic acetylcholine receptors are widely expressed in the nervous system, but their functions remain poorly understood. One attractive hypothesis is that the receptors act presynaptically to modulate synaptic transmission. We provide a direct demonstration of presynaptic nicotinic receptors in situ by using whole-cell patch-clamp techniques to record currents in large presynaptic calyces that midbrain neurons form on ciliary neurons. Bath application of nicotine induced inward currents in the calyces capable of generating action potentials that overrode the limited space clamp achievable. The inward currents reversed near 0 mV and showed inward rectification common for neuronal nicotinic receptors. Tetrodotoxin (TTX) blocked the action potentials but not the inward currents. alpha-Bungarotoxin blocked both, consistent with the presynaptic receptors containing alpha7 subunits. Recording from the postsynaptic ciliary neurons during nicotine exposure revealed EPSCs that TTX blocked, presumably by blocking presynaptic action potentials. The postsynaptic cells also displayed bimodal inward currents caused by their own nicotinic receptors; the bimodal currents were not blocked by TTX but were blocked partially by alpha-bungarotoxin and completely by D-tubocurarine. Dye-filling with Lucifer yellow from the recording pipette confirmed the identity of patched structures and showed no dye transfer between calyx and ciliary neuron. When calyces or ciliary neurons were labeled en mass with neurobiotin and biocytin through nerve roots, dye transfer was rarely observed. Thus, electrical synapses were infrequent and unlikely to influence calyx responses. Immunochemical analysis of preganglionic nerve extracts identified receptors that bind alpha-bungarotoxin and contain alpha7 subunits. The results unambiguously document the existence of functional presynaptic nicotinic receptors.

Novel subpopulation of neuronal acetylcholine receptors among those binding alpha-bungarotoxin.

Neuronal acetylcholine receptors (AChRs) that bind alpha-bungarotoxin (alpha Bgt) (alpha Bgt-AChRs) have previously been found to contain at least one of the alpha 7-alpha 9 gene products. No other gene products of the 11 neuronal AChR genes cloned to date from rat and/or chick have been identified in such receptors. Chick ciliary ganglia have about 20 fmol of alpha Bgt-AChRs that contain alpha 7 subunits and 5 fmol of synaptic-type AChRs that bind the monoclonal antibody (mAb) 35 and collectively contain alpha 3, beta 4, alpha 5, and, to a lesser extent, beta 2 subunits. Using a sensitive solid-phase immunoprecipitation assay, we show here that ciliary ganglia have about 1 fmol of novel putative AChRs that bind both alpha Bgt and mAb 35 but appear to lack all of the known neuronal AChR gene products in ciliary ganglia, including alpha 3, alpha 5, alpha 7, beta 2, and beta 4. The putative receptors are also unlikely to contain either alpha 8 or alpha 9 gene products, because of the known expression patterns of these gene products. Nonetheless, the component sediments at 10 S, as expected for neuronal AChRs, and has a nicotinic pharmacology similar but not identical to that of alpha 7-containing alpha Bgt-AChRs. The AChR alpha 1 gene product expressed in muscle is known to bind both alpha Bgt and mAb 35, and we show here that ciliary ganglia contain small amounts of alpha 1 transcript. The putative ciliary ganglion AChR defined by joint alpha Bgt and mAb 35 binding, however, does not appear to contain alpha 1 subunits. A similar component binding both mAb 35 and alpha Bgt can be detected in sympathetic ganglia and dorsal root ganglia but not in brain, spinal cord, or retina. The developmental time course of the component in ciliary ganglia is comparable to that of the alpha 7-containing alpha Bgt-AChRs. If the component is a functional AChR on ciliary ganglion neurons, as seems likely, it would represent the fourth AChR subtype produced by this population of cells. Our inability to identify subunits comprising the putative receptors raises the possibility that additional AChR genes remain to be cloned.

Neurons can maintain multiple classes of nicotinic acetylcholine receptors distinguished by different subunit compositions.

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.

Expression of neuronal acetylcholine receptor genes in vertebrate skeletal muscle during development.

Of the 15 nicotinic ACh receptor genes identified in vertebrates, only four (alpha 1, beta 1, gamma, and delta) have been shown to be expressed in embryonic skeletal muscle at early times. In mammalian muscle a fifth gene (epsilon) replaces the gamma gene in expression at later times. The remaining 10 nicotinic receptor genes identified to date (alpha 2-alpha 8, beta 2-beta 4) are expressed in the nervous system and are considered neuronal genes. Using RNase protection assays, we show here that four of the neuronal-type genes (alpha 4, alpha 5, alpha 7, and beta 4) are expressed in developing chick skeletal muscle. Two of them (alpha 4 and alpha 7) decline substantially in transcript abundance between embryonic days 11 and 17, as does alpha 1, while the other two (alpha 5 and beta 4) show only moderate decreases over the same time period. At embryonic day 8, alpha 7 transcripts are nearly 20% as abundant as alpha 1 transcripts. In situ hybridizations confirm the presence of alpha 7 transcripts in muscle cells both in cell culture and in embryonic tissue. No evidence was found for expression of the alpha 2, alpha 3, alpha 8, or beta 3 genes in muscle. Immunoprecipitations and immunoblot analysis using subunit-specific monoclonal antibodies reveal alpha 7 protein in muscle, and the amount of protein rises and declines with the amount of alpha 7 mRNA during development. Sucrose gradient analysis demonstrates that the alpha 7 protein is present in muscle as a species of 10S, the size expected for a nicotinic receptor. The alpha 7 species in muscle binds alpha-bungarotoxin but does not contain alpha 1 subunits, indicating that the two kinds of alpha-type gene products segregate during assembly. The results suggest that neuronal AChRs may play a role in early muscle development.

Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes.

A family of genes encoding neuronal acetylcholine receptor (AChR) subunits has been identified and cloned from vertebrates. Expression studies have implied that as few as one or two kinds of subunits may be sufficient to construct neuronal AChRs and that multiple pair-wise combinations of the gene products are capable of generating functional receptors. We show here that a class of AChRs with a predominantly synaptic location on neurons contains receptors having at least three types of subunits and that the subunits are encoded by the alpha 3, beta 4, and alpha 5 AChR genes. In addition, we show that a class of extrasynaptic AChRs on the same neurons contains the alpha 7 subunits but lacks the alpha 3, beta 4, and alpha 5 subunits. The results demonstrate that native AChRs on neurons are more complex in composition than previously appreciated and suggest that constraints on subunit interactions limit the kinds of receptor species produced.

The alpha 5 gene product assembles with multiple acetylcholine receptor subunits to form distinctive receptor subtypes in brain.

The acetylcholine receptor (AChR) alpha 5 gene has been classified as a member of the AChR gene family based on sequence homology. Expression studies, however, have yet to identify a function for the alpha 5 gene product or even to demonstrate an interaction with known AChR subunits. We report here that the alpha 5 gene product is identical to the 49 kd protein previously found on immunoblots of AChRs purified from brain and ciliary ganglia. In brain the alpha 5 gene product is present both in alpha 3- and in alpha 4-based receptor subtypes, while in the ganglion it is found in an alpha 3-based receptor subtype concentrated in postsynaptic membrane. Immunoprecipitation experiments with subunit-specific monoclonal antibodies indicate that some native AChRs are likely to have at least three kinds of subunits, with two being of the alpha type. These findings support new views about the construction of AChRs in neurons.

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure.

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.

Expression of nicotinic acetylcholine receptor subtypes in brain and retina.

Neuronal nicotinic acetylcholine receptors (AChRs) are composed of two types of subunits: ACh-binding (termed alpha 2, alpha 3, alpha 4 ...) and structural (termed beta 2, beta 3, beta 4 ...). AChR subtypes composed of combinations of subunits of these two types encoded by several related genes are expressed in different parts of the nervous system, where they presumably serve different functional roles. Here we identify the ACh-binding subunit of the most prominent chicken brain AChR subtype by N-terminal amino acid sequence and show that it corresponds to the alpha 4 gene. Previously we identified the structural subunit for this AChR subtype from chicken brain as beta 2 by N-terminal amino acid sequence. Thus, this identifies both genes which encode subunits of the major nicotinic AChR subtype in avian brains. By immunoprecipitation, immunohistochemistry, and northern blot analysis we show that alpha 3 (or a very closely related sequence) is expressed at low levels in the brain and relatively high levels in the retina, while alpha 4 is expressed at high levels in the brain and lower levels in the retina. This differential expression indicates that alpha 3-containing 'ganglionic-type' AChRs may be an important AChR subtype in avian retina.

Assembly of Torpedo acetylcholine receptors in Xenopus oocytes.

To study pathways by which acetylcholine receptor (AChR) subunits might assemble, Torpedo alpha subunits were expressed in Xenopus oocytes alone or in combination with beta, gamma, or delta subunits. The maturation of the conformation of the main immunogenic region (MIR) on alpha subunits was measured by binding of mAbs and the maturation of the conformation of the AChR binding site on alpha subunits was measured by binding of alpha-bungarotoxin (alpha Bgt) and cholinergic ligands. The size of subunits and subunit complexes was assayed by sedimentation on sucrose gradients. It is generally accepted that native AChRs have the subunit composition alpha 2 beta gamma delta. Torpedo alpha subunits expressed alone resulted in an amorphous range of complexes with little affinity for alpha Bgt or mAbs to the MIR, rather than in a unique 5S monomeric assembly intermediate species. A previously recognized temperature-dependent failure in alpha subunit maturation may cause instability of the monomeric assembly intermediate and accumulation of aggregated denatured alpha subunits. Coexpression of alpha with beta subunits also resulted in an amorphous range of complexes. However, coexpression of alpha subunits with gamma or delta subunits resulted in the efficient formation of 6.5S alpha gamma or alpha delta complexes with high affinity for mAbs to the MIR, alpha Bgt, and small cholinergic ligands. These alpha gamma and alpha delta subunit pairs may represent normal assembly intermediates in which Torpedo alpha is stabilized and matured in conformation. Coexpression of alpha, gamma, and delta efficiently formed 8.8S complexes, whereas complexes containing alpha beta and gamma or alpha beta and delta subunits are formed less efficiently. Assembly of beta subunits with complexes containing alpha gamma and delta subunits may normally be a rate-limiting step in assembly of AChRs.

TE671 cells express an abundance of a partially mature acetylcholine receptor alpha subunit which has characteristics of an assembly intermediate.

A partially mature form of the nicotinic acetylcholine receptor alpha subunit was found to be expressed in the human cell line TE671. We found that 40-50% of the alpha-bungarotoxin-binding sites in detergent extracts of these cells corresponds to this unassembled alpha subunit. These unassembled alpha subunits are not found in the surface membrane. The unassembled alpha subunits in extracts from TE671 cells appear, like mature receptors, to have a disulfide bond between Cys-192 and Cys-193 near the acetylcholine-binding site. The unassembled alpha subunit binds alpha-bungarotoxin with high affinity, but its dissociation constant is still 5-fold higher than the native assembled acetylcholine receptor. The cholinergic ligands d-tubocurarine and carbamylcholine have negligible affinity for the immature alpha subunit. Similarly, Xenopus oocytes injected with RNA transcripts for the TE671 alpha subunit express an alpha-bungarotoxin-binding component which is insensitive to carbamylcholine and has a sedimentation coefficient on sucrose gradients of 5.0 S. Oocytes injected with RNA for the Torpedo alpha subunit did not have alpha-bungarotoxin binding activity under similar conditions, suggesting a possible differential efficiency in the maturation of this alpha subunit. We examined the binding of monoclonal antibodies specific to the main immunogenic region and found that this epitope on the unassembled alpha subunit was formed, but was not in a fully mature conformation because although these antibodies bound, they bound with lower affinity than to native acetylcholine receptors. Antibodies in myasthenia gravis patient sera also bound to the unassembled alpha subunits, but with an average 14-fold lower titer.

Determination of amino acids critical to the main immunogenic region of intact acetylcholine receptors by in vitro mutagenesis.

The main immunogenic region (MIR) of the acetylcholine receptor (AChR) is the target for the majority of high-affinity autoantibodies produced in myasthenia gravis patients. Some monoclonal antibodies (mAbs) to the MIR bind specifically, but with low affinity, to synthetic AChR alpha subunit peptides with the sequence alpha 67-76. Studies of synthetic peptides suggest that amino acids alpha 68 and alpha 71 may be especially important to the antigenic structure of the MIR. We have studied the contribution of amino acids alpha 68 and alpha 71 to the antigenicity of the MIR on intact AChR by replacing alpha 68 (N) and alpha 71 (D) of Torpedo AChR alpha with alpha 68 (D) and alpha 71 (K) by site-directed mutagenesis, expressing the mutated transcripts in Xenopus oocytes along with wild-type Torpedo beta, gamma and delta subunits, and analyzing the expressed AChR for the binding of mAbs to the MIR. These mutations of the MIR greatly diminished binding of mAbs to the MIR. Thus, both alpha 68 and alpha 71 are critical to the antigenicity of the MIR in intact AChRs.

Brain alpha-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily.

alpha-Bungarotoxin (alpha Bgt) is a potent, high-affinity antagonist for nicotinic acetylcholine receptors (AChRs) from muscle, but not for AChRs from neurons. Both muscle and neuronal AChRs are thought to be formed from multiple homologous subunits aligned around a central cation channel whose opening is regulated by ACh binding. In contrast, the exact structure and function of high-affinity alpha Bgt binding proteins (alpha BgtBPs) found in avian and mammalian neurons remain unknown. Here we show that cDNA clones encoding alpha BgtBP alpha 1 and alpha 2 subunits define alpha BgtBPs as members of a gene family within the ligand-gated ion channel gene superfamily, but distinct from the gene families of AChRs from muscles and nerves. Subunit-specific monoclonal antibodies raised against bacterially expressed alpha BgtBP alpha 1 and alpha 2 subunit fragments reveal the existence of at least two different alpha BgtBP subtypes in embryonic day 18 chicken brains. More than 75% of all alpha BgtBPs have the alpha 1 subunit, but no alpha 2 subunit, and a minor alpha BgtBP subtype (approximately 15%) has both the alpha 1 and alpha 2 subunits.