Adenosine A2A receptor induces protein kinase A-dependent functional modulation of human α3β4 nicotinic receptor (original) (raw)
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Molecular Brain Research, 2003
This study determined whether the a4 subunit of human a4b2 neuronal nicotinic receptors is phosphorylated in situ by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). To accomplish this, human cloned epithelial cells stably transfected 32 with the human a4b2 nicotinic receptor (SH-EP1-ha4b2) were incubated with P-orthophosphate to label endogenous ATP stores, and the phosphorylation of a4 subunits was determined in the absence or presence of PKA or PKC activation. Autoradiographs and immunoblots indicated that a4 subunits immunoprecipitated from a membrane preparation of SH-EP1-ha4b2 cells exhibited a single 32 P-labeled band corresponding to the a4 subunit protein; no signals were associated with untransfected SH-EP1 cells. The a4 subunits from SH-EP1-ha4b2 cells incubated in the absence of the activators exhibited a basal level of phosphorylation that was decreased in the presence of the PKA inhibitor H-89 (5 mM), but unaltered in the presence of the PKC inhibitor Ro-31-8220 (0.1 mM). Activation of PKA by forskolin (10 mM), dibutyryl-cAMP (1 mM), or Sp-8-Br-cAMP (1 mM) enhanced phosphorylation nearly threefold; the inactive isomer, Rp-8-Br-cAMP (1 mM) had no effect. In addition, the forskolin effect was totally blocked by the PKA inhibitor H-89 (5 mM). Activation of PKC by the phorbol esters PDBu (200 nM) or PMA (200 nM) increased a4 subunit phosphorylation approximately twofold, and the PDBu effect was blocked by the selective PKC inhibitor Ro-31-8220 (0.1 mM). These findings indicate that the a4 subunit of human a4b2 nicotinic receptors is phosphorylated in situ by PKA and PKC.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1992
Adenosine modulation of nicotinic ACh receptor (nAChR) function was studied in primary cultures of rat skeletal muscle. Activation of the nAChR by carbachol increased extracellular adenosine concentration in a dose-dependent manner. Furthermore, carbachol activation of the nicotinic receptor resulted in a twofold increase in cAMP levels in the muscle cells. The carbachol-dependent increase in cAMP levels was inhibited by adenosine receptor antagonists as well as by nicotinic receptor antagonists. These results suggest that the increased cAMP levels were due to adenosine receptor activation by the extracellular adenosine accumulated on nAChR activation. Others have shown that desensitization of the nAChR by agonist is mediated, in part, by phosphorylation. Since we found that nicotinic cholinergic agonists also cause adenosine accumulation with concomitant cAMP increases, we determined whether the accumulated adenosine has a role in desensitization. We found that the adenosine recept...
The Journal of Physiology, 2006
Naturally expressed nicotinic acetylcholine receptors (nAChR) containing α4 subunits (α4 * -nAChR) in combination with β2 subunits (α4β2-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. β4 subunits are also richly expressed and colocalize with α4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, α4β4-nAChR also may exist and play important functional roles. In this study, properties were determined of human α4β2-and α4β4-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human α4β4-nAChR have ∼4-fold higher amplitude than those mediated via human α4β2-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at α4β4-nAChR than at α4β2-nAChR. Cytisine and lobeline serve as full agonists at α4β4-nAChR but are only partial agonists at α4β2-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional α4β2-and α4β4-nAChR. Whole-cell current responses show stronger inward rectification for α4β2-nAChR than for α4β4-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR β2 or β4 subunits can combine with α4 subunits to generate two forms of α4 * -nAChR with distinctive physiological and pharmacological features. Diversity in α4 * -nAChR is of potential relevance to nervous system function, disease, and nicotine dependence.
Neuropharmacology, 2014
Nicotinic acetylcholine receptors (nAChRs) containing the a5 subunit are of interest because genomewide association studies and candidate gene studies have identified polymorphisms in the a5 gene that are linked to an increased risk for nicotine dependence, lung cancer, and/or alcohol addiction. To probe the functional impact of an a5 subunit on nAChRs, a method to prepare a homogeneous population of a5-containing receptors must be developed. Here we use a gain of function (9 0 ) mutation to isolate populations of a5-containing nAChRs for characterization by electrophysiology. We find that the a5 subunit modulates nAChR rectification when co-assembled with a4 and b2 subunits. We also probe the a5ea4 interface for possible ligand-binding interactions. We find that mutations expected to ablate an agonist-binding site involving the a5 subunit have no impact on receptor function. The most straightforward interpretation of this observation is that agonists do not bind at the a5ea4 interface, in contrast to what has recently been demonstrated for the a4ea4 interface in related receptors. In addition, our mutational results suggest that the a5 subunit does not replace the a4 or b2 subunits and is relegated to occupying only the auxiliary position of the pentameric receptor.
British Journal of Pharmacology, 2006
The effect of ACh on the release of adenosine was studied in rat whole carotid bodies, and the nicotinic ACh receptors involved in the stimulation of this release were characterized. 2 ACh and nicotinic ACh receptor agonists, cytisine, DMPP and nicotine, caused a concentrationdependent increase in adenosine production during normoxia, with nicotine being more potent and efficient in stimulating adenosine release from rat CB than cytisine and DMPP. 3 D-Tubocurarine, mecamylamine, DHbE and a-bungarotoxin, nicotinic ACh receptor antagonists, caused a concentration-dependent reduction in the release of adenosine evoked by hypoxia. The rank order of potency for nicotinic ACh receptor antagonists that inhibit adenosine release was DHbE4mecamylamine4D-tubocurarine4a-bungarotoxin. 4 The effect of the endogenous agonist, ACh, which was mimicked by nicotine, was antagonized by DHbE, a selective nicotinic receptor antagonist. 5 The ecto-5 0-nucleotidase inhibitor AOPCP produces a 72% inhibition in the release of adenosine from CB evoked by nicotine. 6 Taken together, these data indicate that ACh induced the production of adenosine, mainly from extracellular ATP catabolism at the CB through a mechanism that involves the activation of nicotinic receptors with a4 and b2 receptor subunits.
Neuropharmacology, 2014
A cytosine to thymidine (C / T) missense mutation in the signal peptide (SP) sequence (rs2472553) of the nicotinic acetylcholine receptor (nAChR) a2 subunit produces a threonine-to-isoleucine substitution (T22I) often associated with nicotine dependence (ND). We assessed effects on function of a2*-nAChR ('*'indicates presence of additional subunits) of this mutation, which could alter SP cleavage, RNA/protein secondary structure, and/or efficiency of transcription, translation, subunit assembly, receptor trafficking or cell surface expression. Two-electrode voltage clamp analyses indicate peak current responses to ACh or nicotine are decreased 2.8e5.8-fold for putative low sensitivity (LS; 10:1 ratio of a:b subunit cRNAs injected) a2b2or a2b4-nAChR and increased for putative high sensitivity (HS; 1:10 a:b subunit ratio) a2b2-(5.7e15-fold) or a2b4-(1.9e2.2-fold) nAChR as a result of the mutation. Agonist potencies are decreased 1.6e4-fold for putative LS or HS a2(T22I)b2-nAChR or for either a2*-nAChR subtype formed in the presence of equal amounts of subunit cRNA, slightly decreased for LS a2(T22I)b4-nAChR, but increased 1.4e2.4-fold for HS a2(T22I)b4-nAChR relative to receptors containing wild-type a2 subunits. These effects suggest that the a2 subunit SP mutation generally favors formation of LS receptor isoforms. We hypothesize that lower sensitivity of human a2*-nAChR to nicotine could contribute to increased susceptibility to ND. To our knowledge this is the first report of a SP mutation having a functional effect in a member of cys-loop family of ligand-gated ion channels.
Human α4β2 Neuronal Nicotinic Acetylcholine Receptor in HEK 293 Cells: A Patch-Clamp Study
The Journal of Neuroscience, 1996
The cloning and expression of genes encoding for the human neuronal nicotinic acetylcholine receptors (nAChRs) has opened new possibilities for investigating their physiological and pharmacological properties. Cells (HEK 293) stably transfected with two of the major brain subunits, ␣4 and 2, were characterized electrophysiologically using the patch-clamp technique. Fast application of the natural ligand ACh can evoke currents up to 3500 pA, with an apparent affinity (EC 50) of 3 M and a Hill coefficient of 1.2. The rank order of potency of four nAChR ligands to activate human ␣42 receptors is (Ϫ)-nicotine Ͼ ACh Ͼ (Ϫ)-cytisine Ͼ ABT-418. At saturating concentrations, the efficacy of these ligands is ABT-418 Ͼ Ͼ (Ϫ)-nicotine Ͼ ACh Ͼ Ͼ (Ϫ)-cytisine Ͼ GTS-21 (previously named DMXB). Coapplication of 1 M ACh with known nAChR inhibitors such as dihydro--erythroidine and methyllycaconitine reversibly reduces the current evoked by the agonist with respective IC 50 values of 80 nM and 1.5 M. The current-voltage relationship of human ␣42 displays a strong rectification at positive potentials. Experiments of ionic substitutions suggest that human ␣42 nAChRs are permeable to sodium and potassium ions. In the "outside-out" configuration, ACh evokes unitary currents (main conductance 46 pS) characterized by a very fast rundown. Potentiation of the ACh-evoked currents is observed when the extracellular calcium concentration is increased from 0.2 to 2 mM. In contrast, however, a reduction of the evoked currents is observed when calcium concentration is elevated above 2 mM.