Surface and intracellular distribution of a putative neuronal nicotinic acetylcholine receptor.

Abstract

Abstract. Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the main immunogenic region (MIR) of nicotinic acetylcholine receptors from skeletal muscle and electric organ. Previous studies have shown that the component has many of the properties expected for a ganglionic nicotinic acetylcholine receptor, and that its distribution on the neuron surface in vivo is restricted predominantly to synaptic membrane. Here we report the presence of a large intracellular pool of the putative receptor in embryonic neurons and demonstrate that it is associated with organelles known to comprise the biosynthetic and regulatory pathways of integral plasma membrane proteins. Embryonic chick ciliary ganglia were lightly fixed, saponinpermeabilized, incubated with an anti-MIR monoclonal antibody (mAb) followed by horseradish peroxidase-conjugated secondary antibody, reacted for peroxidase activity, and examined by electron microscopy. Deposits of reaction product were associated with synaptic membrane, small portions of the pseudodendrite surface membrane, most of the rough endoplasmic reticulum, small portions of the nuclear envelope, some Golgi complexes, and a few coated pits, coated vesicles, multivesicular bodies, and smooth-membraned vacuoles. No other labeling was present in the neurons. The labeling was specific in that it was not present when the anti-MIR mAb was replaced with either nonimmune serum or mAbs of different specificity. Chick dorsal root ganglion neurons thought to lack nicotinic acetylcholine receptors were not labeled by the anti-MIR mAb. Substantial intracellular populations have also been reported for the muscle acetylcholine receptor and brain voltagedependent sodium channel ct-subunit. This may represent a general pattern for multisubunit membrane proteins during development.ITTLE is known about the distribution and regulation of nicotinic acetylcholine receptors (AChRs) 1 on neurons because methods have not been available for unambiguously identifying the receptor protein. Chick ciliary ganglion neurons have nicotinic AChRs that mediate chemical synaptic transmission through the ganglion (Martin and Pilar, 1963a, b). Recently monoclonal antibodies (mAbs) to the main immunogenic region (MIR) of AChR ¢t-subunit from muscle and electric organ have been used to identify a membrane component on chick ciliary ganglion neurons that has many of the properties expected for a neuronal nicotinic AChR. On the neuron surface, the cross-reacting component is located predominantly in synaptic membrane (Jacob et al., 1984). It is an integral membrane component that in detergent extracts has a sedimentation coefficient similar to AChR from muscle and electric organ; it binds to concanavalin A, a lectin known to block AChR function on 1. Abbreviations used in this paper: AChR, acetylcholine receptor; HRP, horseradish peroxidase; mAb, monoclonal antibody; MIR, main immunogenic region; PBS-glycine, phosphate-buffered saline containing 0.75 % glycine; PG, phosphate-buffered...

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

“…This preparation, therefore, provides a valuable opportunity to compare molecular mechanisms that orchestrate the assembly of the different receptor complexes. The α3*nAChRs are concentrated in postsynaptic membrane regions that oppose presynaptic terminal active zones (Jacob et al, 1986;Williams et al, 1998). GlyR clusters localize to separate but proximal postsynaptic membrane regions, all under one presynaptic terminal (Tsen et al, 2000).…”

Regulatory mechanisms that govern nicotinic synapse formation in neurons

“…In particular, the ␣-bgt-sensitive receptors have higher calcium permeability, and in some neuron populations faster kinetics of activation and desensitization, but a slowly desensitizing response in others (Seguela et al, 1993;Ullian et al, 1997;Chang and Berg, 1999;Cuevas et al, 2000). Within one neuron, the diverse nAChR subtypes are spatially segregated relative to one another and target to discrete synapse-associated sites: the presynaptic terminal, the specialized postsynaptic membrane, and the perisynaptic dendritic surface membrane (Jacob and Berg, 1983;Jacob et al, 1984Jacob et al, , 1986Loring et al, 1985;Loring and Zigmond, 1987;Moss and Role, 1993;Horch and Sargent, 1995;McGehee and Role, 1995;Gray et al, 1996;Shoop et al, 1999). The spatial segregation and distinct biophysical properties of the diverse nAChR subtypes are likely to create functionally specialized synapse-associated microregions and establish distinct spatial and temporal patterns of calcium influx that locally target different downstream signaling events (see review by D.K.…”

“…Individual CG neurons express two distinct types of nAChRs: ␣3-nAChRs and ␣7-nAChRs. The ␣3-nAChRs, composed of ␣3, ␣5, and ␤4 (occasionally ␤2) subunits, are largely concentrated in the postsynaptic membrane (Jacob et al, 1986;Loring and Zigmond, 1987;Vernallis et al, 1993;Horch and Sargent, 1995). In contrast, the ␣7-nAChRs, composed of ␣7 subunits, are excluded from the synapse and restricted to perisynaptic regions on somatic spines (Jacob and Berg, 1983;Loring et al, 1985;Vernallis et al, 1993;Horch and Sargent, 1995;Shoop et al, 1999;Conroy and Berg, 2000).…”

“…Although ␣3-nAChR and ␣7-nAChR total protein levels are reduced in input-deprived CG neurons, whole-cell ACh responses are unchanged relative to age-matched controls (Engisch and Fischbach, 1992;Levey et al, 1995). The absence of a change in functional nAChR surface levels, despite declines in nAChR mRNA and protein levels, can possibly be explained by the observation that only a very small proportion (approximately 5%) of the internal biosynthetic pool of nAChRs is destined for the surface in developing CG neurons (Jacob et al, 1986;Stollberg and Berg, 1987;Levey et al, 1995). These data suggest that the mechanisms that govern nAChR surface expression on neurons do not require innervation, and that other positive regulatory signals are active in the complex in vivo cellular environment (see below).…”

“…Two pools of nAChRs are found in CG neurons: a surface membrane pool and a larger internal pool that is associated with organelles that function in the biosynthesis, processing, and transport of integral membrane proteins (Jacob et al, 1986;Stollberg and Berg, 1987;Jacob, 1991). Studies of the regulatory effects of presynaptic innervation (detailed above) demonstrate that these two pools can be independently regulated.…”