William G. Conroy

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 α5 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.

Three subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors are expressed in chick retina

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.

PDZ-Containing Proteins Provide a Functional Postsynaptic Scaffold for Nicotinic Receptors in Neurons

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.

Developmental expression of nicotinic receptors in the chick and human spinal cord

Naturally occurring programmed cell death of lumbar motor neurons in the chick spinal cord occurs between embryonic day (E) 6 and E12; whereas, a peak of motor neuron degeneration in the human spinal cord occurs between 12 and 16 weeks gestation. One of the major neurotransmitters, acetylcholine, is released from the embryonic motor neuron early in development and is thought to be responsible for early muscle activity that serves as a signal for regulating motor neuron survival. The effects of acetylcholine are mediated by two functionally distinct classes of receptors; namely, muscarinic and nicotinic with nicotinic receptors being used at the neuromuscular synapse. In this study, we determined the developmental expression profile of nicotinic acetylcholine receptor subunits in the chick and human lumbar motor neurons and skeletal muscle using reverse transcription polymerase chain reaction, immunoblots, and immunocytochemistry. Our results show that, in the chick, nicotinic receptor subunits α1, α4, α7, α8, and β2 appear to be regulated during the process of naturally occurring motor neuron cell death in the spinal cord. A new finding was the expression of α8 mRNA and protein from E4.5 through E7 in chick motor neurons. Interestingly, we also found that, at E14, α8 protein was localized only in sensory dorsal horn neurons. In the developing human spinal cord, we determined that nicotinic receptor subunits α1, α2, α3, α4, α7, β2, and β3 were expressed before the programmed cell death period, and α2, α4, α7, β2, β3, and β4 were expressed during the programmed cell death period. Our data demonstrate that neuronal and muscle nicotinic receptor mRNAs and proteins are expressed during important embryonic periods. This finding raises the possibility that nicotinic receptors play an important role in the spinal cord and skeletal muscle during early development. J. Comp. Neurol. 455:86–99, 2003.

EphB receptors co-distribute with a nicotinic receptor subtype and regulate nicotinic downstream signaling in neurons.

Activation of nicotinic acetylcholine receptors (nAChRs) on neurons engages calcium-dependent signaling pathways regulating numerous events. Receptors containing alpha7 subunits (alpha7-nAChRs) are prominent in this because of their abundance and high relative calcium permeability. We show here that EphB2 receptors are co-localized with postsynaptic alpha7-nAChRs on chick ciliary ganglion neurons and that treatment of the cells with an ephrinB1 construct to activate the EphB receptors exerts physical restraints on both classes of receptors, diminishing their dispersal after spine retraction or lipid raft disruption. Moreover, the ephrinB1/EphB receptor complex specifically enhances the ability of alpha7-nAChRs to activate the transcription factor CREB, acting through a pathway including a receptor tyrosine kinase, a Src family member, PI3 kinase, and protein kinase A most distally. The enhancement does not appear to result from a change in the alpha7-nAChR current amplitude, suggesting a downstream target. The results demonstrate a role for ephrin/EphB action in nicotinic signaling.

Synchronous and Asynchronous Transmitter Release at Nicotinic Synapses Are Differentially Regulated by Postsynaptic PSD-95 Proteins

The rate and timing of information transfer at neuronal synapses are critical for determining synaptic efficacy and higher network function. Both synchronous and asynchronous neurotransmitter release shape the pattern of synaptic influences on a neuron. The PSD-95 family of postsynaptic scaffolding proteins, in addition to organizing postsynaptic components at glutamate synapses, acts transcellularly to regulate synchronous glutamate release. Here we show that PSD-95 family members at nicotinic synapses on chick ciliary ganglion neurons in culture execute multiple functions to enhance transmission. Together, endogenous PSD-95 and SAP102 in the postsynaptic cell appear to regulate transcellularly the synchronous release of transmitter from presynaptic terminals onto the neuron while stabilizing postsynaptic nicotinic receptor clusters under the release sites. Endogenous SAP97, in contrast, has no effect on receptor clusters but acts transcellularly from the postsynaptic cell through N-cadherin to enhance asynchronous release. These separate and parallel regulatory pathways allow postsynaptic scaffold proteins to dictate the pattern of cholinergic input a neuron receives; they also require balancing of PSD-95 protein levels to avoid disruptive competition that can occur through common binding domains.

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. J. Comp. Neurol. 425:284–294, 2000.

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

Abstract : Nicotinic acetylcholine receptors (nAChRs) containing the α7 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 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 α7‐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.