Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture

Abstract

Chick ciliary ganglion neurons have nicotinic ACh receptors that mediate synaptic input to the cells. Ultrastructural studies with a monoclonal antibody that recognizes the neuronal ACh receptor have previously shown that, in addition to a predominantly synaptic location for the receptors on the neuron surface in vivo, substantial amounts of intracellular receptor are present as well. Here we report that intracellular receptor and smaller receptor-related components make up at least two-thirds of the total antibody binding sites associated with the ciliary ganglion neurons in cell culture. The intracellular sites for the most part represent integral membrane components that bind to concanavalin A when solubilized, indicating that the components are glycosylated. Sucrose gradient analysis shows that the intracellular material includes a 10 S component, likely to represent assembled receptor, along with species sedimenting in the 5–9 S range. Blocking the surface sites with unlabeled antibody and measuring the appearance of the new receptor on the cell surface with radiolabeled antibody indicates that the receptors are inserted into the plasma membrane at a rate equivalent to about 4% of the total surface receptor per hour. The transit time for newly synthesized receptor to reach the cell surface appears to be 2–3 hr. These observations suggest that about 5% of the intracellular receptors are transported to the cell surface in culture. The half-life of ACh receptors in the plasma membrane was estimated by 2 different approaches to be about 22 hr. Surface and internal sites respond in a qualitatively similar way to external agents that specifically modulate cholinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Native α3β4* Neuronal Nicotinic Receptors: Binding and Functional Studies Investigating Turnover of Surface and Intracellular Receptor Populations

“…The data presented here suggest that the intracellular pool of receptors does not play a primary role in recovery after alkylation or in normal turnover because new receptor synthesis is required in both cases. These findings are however consistent with previous studies that suggest only a small percentage of the intracellular binding sites in chick ciliary ganglion neuron (Stollberg and Berg, 1987) or muscle cells (Pestronk, 1985) are destined Fig. 7.…”

“…These intracellular nAChRs may consist of two distinct pools: nAChRs destined for immediate surface expression and nAChRs stored as an independent pool. Intracellular nAChR pools in other tissues (Stollberg and Berg, 1987) and recombinant cell lines (Whiteaker et al, 1998) can be modulated (Rothhut et al, 1996) and may be involved with the receptor turnover process. It is also possible that these intracellular nAChRs may represent internalized receptors that were once on the receptor surface.…”

Rapid Activity-Driven SNARE-Dependent Trafficking of Nicotinic Receptors on Somatic Spines

“…Several hours are required for constitutive receptor synthesis, transport, and insertion into the plasma membrane. Agonist-induced downregulation of nicotinic receptors also occurs over hours and involves a significant reduction in the complement of receptors on the neuron surface (Messing, 1982;Stollberg and Berg, 1987). At the neuromuscular junction, receptor blockade can accelerate the rate of nicotinic receptor removal, but both the mechanism and time course differ significantly from that seen here for ␣7-nAChRs (Akaaboune et al, 1999).…”

Regulatory mechanisms that govern nicotinic synapse formation in neurons

“…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.…”