Effects of chronic nicotine on heteromeric neuronal nicotinic receptors in rat primary cultured neurons

Nicotine increases the number of neuronal nicotinic acetylcholine receptors (nAChRs) in brain. This study investigated the effects of chronic nicotine treatment on nAChRs expressed in primary cultured neurons. In particular, we studied the chronic effects of nicotine exposure on the total density, surface expression and turnover rate of heteromeric nAChRs. The receptor density was measured by [12?I]epibatidine ([12?I]EB) binding. Untreated and nicotine-treated neurons were compared from several regions of embryonic (E19) rat brain. Twelve days of treatment with 10 μM nicotine produced a twofold up-regulation of nAChRs. Biotinylation and whole-cell binding studies indicated that up-regulation resulted from an increase in the number of cell surface receptors as well as intracellular receptors. nAChR subunit composition in cortical and hippocampal neurons was assessed by immunoprecipitation with subunit-selective antibodies. These neurons contain predominantly α4, β2 and α5 subunits, but α2, α3, α6 and β4 subunits were also detected. Chronic nicotine exposure yielded a twofold increase in the β2-containing receptors and a smaller up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover rate of surface receptors was > 2 weeks and chronic nicotine exposure had no effect on this rate.     

Tyrosine kinase inhibitors alter composition of nicotinic receptors on neurons

Protein tyrosine kinase (PTK) inhibitors were used to examine the roles of tyrosine phosphorylation in synaptic function. We show here that two different PTK inhibitors, herbimycin A and lavendustin A, both selectively downregulate a subpopulation of nicotinic acetylcholine receptors (AChRs) on chick ciliary ganglion neurons in culture. The downregulation requires a number of hours to occur and involves only those receptors containing the α3, α5, and β4 gene products. Not affected are AChRs that additionally contain the β2 gene product or AChRs that are made up of the α7 gene product. The downregulation preferentially targets receptors destined for the cell surface and has little effect on the large pool of intracellular receptors. The receptor loss is not additive with that seen in the presence of either cycloheximide or tunicamycin, two compounds that the block appearance of new receptors. The downregulation induced by herbimycin A in surface receptors is accompanied by a specific decrement in the amount of α3 protein in the cells. The results indicate that PTKs, either by phosphorylating AChR gene products directly or by acting through intermediary proteins, regulate the size and composition of the AChR pool maintained on the cell surface. Receptor regulation by PTKs may provide a mechanism for long-term control of synaptic signaling between neurons. © 1996 John Wiley & Sons, Inc.     

An extracellular RRR motif flanking the M1 transmembrane domain governs the biogenesis of homomeric neuronal nicotinic acetylcholine receptors

We have previously demonstrated that the highly conserved R209, that flanks the M1 transmembrane segment of nicotinic acetylcholine (ACh) receptors, is required for the transport of assembled homomeric neuronal α7 nicotinic ACh receptors to the cell surface. In the present paper we show that basic residues at positions 208 and 210 are necessary for the assembly of α7 receptors. On the contrary, a basic residue at position 210 of α3 subunit decreases the assembly of heteromeric neuronal α3β4 nicotinic ACh receptors. A basic residue at position 210 of the β4 subunit slightly decreases α3β4 receptor expression. We conclude that a pre-M1 RRR motif is necessary for the biogenesis of homomeric α-bungarotoxin-sensitive neuronal α7 nicotinic ACh receptors.     

Role of protein phosphatases in insulin-like growth factor II (IGF II)-stimulated mannose 6-phosphate/IGF II receptor redistribution.

The regulated expression of mannose 6-phosphate/insulin-like growth factor II (M6P/IGF II) receptors in plasma membranes has previously been shown to be accompanied by marked changes in the phosphorylation state of the receptors (Corvera, S., Folander, K., Clairmont, K. B., and Czech, M. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7567-7571). In the present study we show that protein phosphatase 2A dephosphorylates the human M6P/IGF II receptor in vitro. Incubation of human fibroblasts with okadaic acid, a specific inhibitor of this phosphatase, resulted in a depletion of M6P/IGF II receptors at the cell surface without affecting their internalization kinetics. The phosphorylation state of the remaining cell surface receptors was 3-fold increased. Thus, the endocytosis rate of M6P/IGF II receptors appears to be unaltered by increased phosphorylation. While the decreased cell surface expression of receptors was reversible upon removal of okadaic acid the IGF II-induced redistribution of M6P/IGF II receptors to the plasma membrane (Braulke, T., Tippmer, S., Neher, E., and von Figura, K. (1989) EMBO J. 8, 681-686) was irreversibly inhibited by the phosphatase inhibitor. Receptor redistribution in response to protein kinase C activation was not affected by okadaic acid. These results suggest that the cell surface expression of M6P/IGF II receptor can be regulated by phosphatase-dependent and -independent pathways. In addition, the phosphorylation state and the steady-state cell surface number of transferrin receptors were not affected by okadaic acid, whereas it impaired the IGF II-stimulated receptor redistribution similarly as for M6P/IGF II receptors. The data indicate that okadaic acid-sensitive protein phosphatases may play a general role in terms of IGF II-modulated receptor recycling.     

A model for the assembly of nicotinic receptors based on subunit-subunit interactions

Neuronal ion-channels are complex multimeric proteins. Within a given family, the variability of their pharmacological responses depends on subunit composition and subunit arrangement. We report here that protein assembly in the pentameric nicotinic acetylcholine receptor family, the best characterized of all neuronal receptors, can be predicted using information derived from homology modeled surface to surface subunit interactions based on the atomic structure of a snail acetylcholine-binding protein. An empirical assembly model is able to establish both subunit stoichiometry and subunit arrangement of known neuronal and muscle nicotinic receptors. This contribution to the understanding of nicotinic receptor assembly and variability might be extended to other types of ion-channels.     

Natural α-conotoxins and their synthetic analogues in study of nicotinic acetylcholine receptors

α-Conotoxins, peptide neurotoxins from poisonous marine snails of the genus Conus that highly specifically block nicotinic acetylcholine receptors (AChRs) of various types, are reviewed. Preliminarily, the structural organization of AChRs of the muscular and neuronal types, their involvement in physiological processes, and their role in various diseases are briefly discussed. In this connection, the necessity of quantitative determination of AChR subtypes using neurotoxins and other approaches is substantiated. The chemical structure, spatial organization, and specificity of α-conotoxins are mainly discussed, taking into consideration the recent results on the ability of α-conotoxins to interact with muscular or neuronal hetero-and homooligomeric AChRs exhibiting a high species specificity. Particular emphasis is placed upon a thorough characterization of the surfaces of interaction of α-conotoxins with AChRs using synthetic analogues of α-conotoxins, mutations in AChRs, and pairwise mutations in both α-conotoxins and AChRs. The discovery in 2001 of the acetylcholine-binding protein from the pond snail Lymnaea stagnalis and the determination of its crystalline structure led to rapid progress in understanding the structural organization of ligand-binding domains of AChRs with which α-conotoxins also interact. We discuss the interaction of various α-conotoxins with acetylcholine-binding proteins, the recently reported X-ray structure of the complex of the acetylcholine-binding protein from Aplysia californica with the α-conotoxin analogue PnIA, and the application of this structure to the modeling of complexes of α-conotoxins with various AChRs.     

Efficiency of acetylcholine receptor subunit assembly and its regulation by cAMP

Assembly of nicotinic acetylcholine receptor (AChR) subunits was investigated using mouse fibroblast cell lines stably expressing either Torpedo (All-11) or mouse (AM-4) alpha, beta, gamma, and delta AChR subunits. Both cell lines produce fully functional cell surface AChRs. We find that two independent treatments, lower temperature and increased intracellular cAMP can increase AChR expression by increasing the efficiency of subunit assembly. Previously, we showed that the rate of degradation of individual subunits was decreased as the temperature was lowered and that Torpedo AChR expression was acutely temperature sensitive, requiring temperatures lower than 37 degrees C. We find that Torpedo AChR assembly efficiency increases 56-fold as the temperature is decreased from 37 to 20 degrees C. To determine how much of this is a temperature effect on degradation, mouse AChR assembly efficiencies were determined and found to be only approximately fourfold more efficient at 20 than at 37 degrees C. With reduced temperatures, we can achieve assembly efficiencies of Torpedo AChR in fibroblasts of 20-35%. Mouse AChR in muscle cells is also approximately 30% and we obtain approximately 30% assembly efficiency of mouse AChR in fibroblasts (with reduced temperatures, this value approaches 100%). Forskolin, an agent which increases intracellular cAMP levels, increased subunit assembly efficiencies twofold with a corresponding increase in cell surface AChR. Pulse-chase experiments and immunofluorescence microscopy indicate that oligomer assembly occurs in the ER and that AChR oligomers remain in the ER until released to the cell surface. Once released, AChRs move rapidly through the Golgi membrane to the plasma membrane. Forskolin does not alter the intracellular distribution of AChR. Our results indicate that cell surface expression of AChR can be regulated at the level of subunit assembly and suggest a mechanism for the cAMP-induced increase in AChR expression.     

Activity-dependent phosphorylation of GABAA receptors regulates receptor insertion and tonic current

The expression of GABA(A) receptors and the efficacy of GABAergic neurotransmission are subject to adaptive compensatory regulation as a result of changes in neuronal activity. Here, we show that activation of L-type voltage-gated Ca(2+) channels (VGCCs) leads to Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of S383 within the β3 subunit of the GABA(A) receptor. Consequently, this results in rapid insertion of GABA(A) receptors at the cell surface and enhanced tonic current. Furthermore, we demonstrate that acute changes in neuronal activity leads to the rapid modulation of cell surface numbers of GABA(A) receptors and tonic current, which are critically dependent on Ca(2+) influx through L-type VGCCs and CaMKII phosphorylation of β3S383. These data provide a mechanistic link between activity-dependent changes in Ca(2+) influx through L-type channels and the rapid modulation of GABA(A) receptor cell surface numbers and tonic current, suggesting a homeostatic pathway involved in regulating neuronal intrinsic excitability in response to changes in activity.     

GABRB3 mutation, G32R, associated with childhood absence epilepsy alters α1β3γ2L γ-aminobutyric acid type A (GABAA) receptor expression and channel gating

A GABA(A) receptor β3 subunit mutation, G32R, has been associated with childhood absence epilepsy. We evaluated the possibility that this mutation, which is located adjacent to the most N-terminal of three β3 subunit N-glycosylation sites, might reduce GABAergic inhibition by increasing glycosylation of β3 subunits. The mutation had three major effects on GABA(A) receptors. First, coexpression of β3(G32R) subunits with α1 or α3 and γ2L subunits in HEK293T cells reduced surface expression of γ2L subunits and increased surface expression of β3 subunits, suggesting a partial shift from ternary αβ3γ2L receptors to binary αβ3 and homomeric β3 receptors. Second, β3(G32R) subunits were more likely than β3 subunits to be N-glycosylated at Asn-33, but increases in glycosylation were not responsible for changes in subunit surface expression. Rather, both phenomena could be attributed to the presence of a basic residue at position 32. Finally, α1β3(G32R)γ2L receptors had significantly reduced macroscopic current density. This reduction could not be explained fully by changes in subunit expression levels (because γ2L levels decreased only slightly) or glycosylation (because reduction persisted in the absence of glycosylation at Asn-33). Single channel recording revealed that α1β3(G32R)γ2L receptors had impaired gating with shorter mean open time. Homology modeling indicated that the mutation altered salt bridges at subunit interfaces, including regions important for subunit oligomerization. Our results suggest both a mechanism for mutation-induced hyperexcitability and a novel role for the β3 subunit N-terminal α-helix in receptor assembly and gating.     

alpha 7-type acetylcholine receptor localization and its modulation by nicotine and cholesterol in vascular endothelial cells

The neuronal‐type α7 nicotinic acetylcholine receptor (α7AChR) is also found in various non‐neural tissues, including vascular endothelium, where its peculiar ionotropic properties (high Ca²⁺ permeability) and its supervening Ca²⁺‐mediated intracellular cascades may play important roles in physiology (angiogenesis) and pathology (inflammation and atherogenesis). Changes in molecular (up‐regulation, affinity, and conformational states) and cellular (distribution, association with membranes) properties of the α7AChR related to angiogenesis (wound‐repair cell migration) and atherogenesis (alterations in cholesterol content) were studied in living endothelial cells, with the aim of determining whether such changes constitute early markers of inflammatory response. The combination of pharmacological, biochemical, and fluorescence microscopy tools showed that α7AChRs in rat arterial endothelial (RAEC) and human venous endothelial (HUVEC) cells occur at extremely low expression levels (～50 fmol/mg protein) but undergo agonist‐induced up‐regulation at relatively high nicotine concentrations (～300‐fold with 50 µM ligand), increasing their cell‐surface exposure. When analyzed in terms of cold Triton X‐100 solubility and subcellular distribution, α7AChRs occur in the “non‐raft” subcellular membrane fractions. Acute cholesterol depletion reduced not only cholesterol levels but also the number of cell‐surface α7AChRs. Nicotine exposure markedly stimulated cell migration and accelerated wound repair, which drastically diminished in cells deprived of the sterol. The angiogenic effect of nicotine appears to be synergistic with cholesterol content. Finally, the apparent K_D of α7AChRs for the open‐channel blocker crystal violet was found to be ～600‐fold lower in receptor‐enriched membranes obtained from up‐regulated HUVEC. J. Cell. Biochem. 112: 3276–3288, 2011. © 2011 Wiley Periodicals, Inc.     

Acetylcholine receptors are required for agrin-induced clustering of postsynaptic proteins.

We have investigated the role of acetylcholine receptors (AChRs) in an early step of postsynaptic assembly at the neuromuscular synapse, the clustering of postsynaptic proteins induced by nerve-released agrin. To achieve this, we used two variants of C2 myotubes virtually lacking AChRs and C2 cells in which surface AChRs were down-regulated by AChR antibodies. In all cases, agrin caused normal clustering of the agrin receptor component MuSK, alpha-dystrobrevin and utrophin, but failed to aggregate AChRs, alpha- and beta-dystroglycan, syntrophin isoforms and rapsyn, an AChR-anchoring protein necessary for postsynaptic assembly and AChR clustering. In C2 variants, the stability of rapsyn was decreased, whereas in antibody-treated cells, rapsyn efficiently co-localized with remaining AChRs in microaggregates. Upon ectopic injection into myofibers in vivo, rapsyn did not form clusters in the absence of AChRs. These results show that AChRs and rapsyn are interdependent components of a pre-assembled protein complex that is required for agrin-induced clustering of a full set of postsynaptic proteins, thus providing evidence for an active role of AChRs in postsynaptic assembly.     

Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, γ-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from α4β2/3 with or without the δ subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the α4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the β3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the δ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of α4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the α4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of α4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the α4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.     

Role of non-neuronal nicotinic acetylcholine receptors in angiogenesis

Angiogenesis is a critical physiological process for cell survival and development. Endothelial cells, necessary for the course of angiogenesis, express several non-neuronal nicotinic acetylcholine receptors (AChRs). The most important functional non-neuronal AChRs are homomeric α7 AChRs and several heteromeric AChRs formed by a combination of α3, α5, β2, and β4 subunits, including α3β4-containing AChRs. In endothelial cells, α7 AChR stimulation indirectly triggers the activation of the integrin αvβ3 receptor and an intracellular MAP kinase (ERK) pathway that mediates angiogenesis. Non-selective cholinergic agonists such as nicotine have been shown to induce angiogenesis, enhancing tumor progression. Moreover, α7 AChR selective antagonists such as α-bungarotoxin and methyllycaconitine as well as the non-specific antagonist mecamylamine have been shown to inhibit endothelial cell proliferation and ultimately blood vessel formation. Exploitation of such pharmacologic properties can lead to the discovery of new specific cholinergic antagonists as anti-cancer therapies. Conversely, the pro-angiogenic effect elicited by specific agonists can be used to treat diseases that respond to revascularization such as diabetic ischemia and atherosclerosis, as well as to accelerate wound healing. In this mini-review we discuss the pharmacological evidence supporting the importance of non-neuronal AChRs in angiogenesis. We also explore potential intracellular mechanisms by which α7 AChR activation mediates this vital cellular process.     

PKCγ is required for ethanol-induced increases in GABA(A) receptor α4 subunit expression in cultured cerebral cortical neurons

Ethanol exposure produces alterations in GABA(A) receptor function and expression associated with CNS hyperexcitability, but the mechanisms of these effects are unknown. Ethanol is known to increase both GABA(A) receptor α4 subunits and protein kinase C (PKC) isozymes in vivo and in vitro. Here, we investigated ethanol regulation of GABA(A) receptor α4 subunit expression in cultured cortical neurons to delineate the role of PKC. Cultured neurons were prepared from rat pups on postnatal day 0-1 and tested after 18?days. GABA(A) receptor α4 subunit surface expression was assessed using P2 fractionation and surface biotinylation following ethanol exposure for 4?h. Miniature inhibitory post-synaptic currents were measured using whole cell patch clamp recordings. Ethanol increased GABA(A) receptor α4 subunit expression in both the P2 and biotinylated fractions, while reducing the decay time constant in miniature inhibitory post-synaptic currents, with no effect on γ2 or δ subunits. PKC activation mimicked ethanol effects, while the PKC inhibitor calphostin C prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression. PKCγ siRNA knockdown prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression, but inhibition of the PKCβ isoform with PKCβ pseudosubstrate had no effect. We conclude that PKCγ regulates ethanol-induced alterations in α4-containing GABA(A) receptors.     

Involvement of calpains in the destabilization of the acetylcholine receptor clusters in rat myotubes

The effects of calpain inhibitors on the total number of acetylcholine receptors (AChRs) on cultured rat myotubes and on the stability of AChR clusters in these myotubes were investigated. The degradation rate of total AChRs labeled with (125)I-alpha-bungarotoxin was assessed from radioactivity remaining in the myotubes as a function of time. Treatment with calpain inhibitors resulted in a two- to three-fold increase in the half-life of total AChRs. Incubation with these inhibitors produced 40% increases in intracellular AChRs but no major changes in surface AChRs, indicating that the increased AChR half-life is due to intracellular accumulation. The rate loss of AChRs from the clusters was assessed by measuring the loss of fluorescence intensity in rhodaminated-alpha-bungarotoxin-labeled clusters with time. Treatment with calpain inhibitors resulted in twofold increases in cluster half-life. Thus, there was generally no change in total surface receptors with the calpain inhibitors, whereas cluster half-life was substantially increased. Furthermore, with a low dose of calpeptin there was no change in turnover of total cellular AChRs, whereas cluster half-life was doubled. Taken together, these results suggest that the increased half-life of clusters produced by the calpain inhibitors may be due to retardation of the lateral movement from AChRs in the clusters.     

ACETYLCHOLINE RECEPTORS: NUMBER and DISTRIBUTION IN INTACT and DEAFFERENTED SUPERIOR CERVICAL GANGLION OF THE RAT1

Abstract— α-Bungarotoxin (α-BuTX) has been used as a marker for studying the acetylcholine receptors (AChRs) in the superior cervical ganglion (SCG) of the adult rat. Binding of [¹²⁵I]α-BuTX to detergent-solubilized AChRs from rat SCG is a saturable and practically irreversible process. The rate constant of association of the toxin-receptor complex is 1.66 × 10⁵M ^?1 S^?1. The receptor is of nicotinic type. One SCG of adult rat binds about 57 fmol of [¹²⁵I]α-BuTX corresponding to 9.2 × 10⁵ AChRs per sympathetic neuron. Light microscope autoradiography shows that AChRs are mainly localized along neuronal processes (probably dendrites). The perikarya exhibit a weak radioactive reaction, while the nerve fibres are devoid of AChRs. Following preganglionic denervation the number of AChRs never increases and their spatial distribution seems not to change.     

α9β1 Integrin in melanoma cells can signal different adhesion states for migration and anchorage

Cell surface integrins are the primary receptors for cell migration on extracellular matrix, and exist in several activation states regulated in part by ectodomain conformation. The α9 integrin subunit, which pairs only with β1, has specific roles in the immune system and may regulate cell migration. Melanoma cells express abundant α9β1 integrin, and its role in cell migration was assessed. Ligands derived from Tenascin-C and ADAM12 supported α9β1 integrin-mediated cell attachment and GTP-Rac dependent migration, but not focal adhesion formation. Manganese ions induced α9β1 integrin- and Rho kinase-dependent focal adhesion and stress fibre formation, suggesting that the activation status of α9β1 integrin was altered. The effect of manganese ions in promoting focal adhesion formation was reproduced by β1 integrin activating antibody. The α9β1 integrin translocated to focal adhesions, where active β1 integrin was also detected by conformation-specific antibodies. Focal adhesion assembly was commensurate with reduced cell migration. Endogenous α9β1 integrin-mediated adhesion was sensitive to the PP1 chemical inhibitor and an inhibitor of endosomal vesicle recycling, but not inhibitors of protein kinase C or the small GTPase Rho. Our results demonstrated that although α9β1 integrin can induce and localise to focal adhesions in a high activation state, its intermediate activity state normally supports cell adhesion consistent with migration.