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1.
Int J Mol Sci ; 23(16)2022 Aug 14.
Article in English | MEDLINE | ID: mdl-36012367

ABSTRACT

The heteromeric assembly of α3 and ß4 subunits of acetylcholine nicotinic receptors (nAChRs) seems to mediate the secretory response in bovine chromaffin cells. However, there is no information about the localization of these nAChRs in relationship with the secretory active zones in this cellular model. The present work presents the first evidence that, in fact, a population of these receptors is associated through the F-actin cytoskeleton with exocytotic machinery components, as detected by SNAP-25 labeling. Furthermore, we also prove that, upon stimulation, the probability to find α3ß4 nAChRs very close to exocytotic events increases with randomized distributions, thus substantiating the clear dynamic behavior of these receptors during the secretory process. Modeling on secretory dynamics and secretory component distributions supports the idea that α3ß4 nAChR cluster mobility could help with improving the efficiency of the secretory response of chromaffin cells. Our study is limited by the use of conventional confocal microscopy; in this sense, a strengthening to our conclusions could come from the use of super-resolution microscopy techniques in the near future.


Subject(s)
Chromaffin Cells , Receptors, Nicotinic , Acetylcholine , Animals , Biological Transport , Cattle , Chromaffin Cells/metabolism , Nicotinic Antagonists , Receptors, Nicotinic/metabolism
2.
Int J Mol Sci ; 22(2)2021 Jan 19.
Article in English | MEDLINE | ID: mdl-33478095

ABSTRACT

The α7 nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain, and inflammation. Allosteric modulation of this receptor might be advantageous to reduce the toxicity in comparison with full agonists. Our previous results obtained with some hydroxy-chalcones, which were identified as positive allosteric modulators (PAMs) of α7 nAChR, prompted us to evaluate the potential of some structurally related naturally occurring flavonoids and curcuminoids and some synthetic curcumin analogues, with the aim of identifying new allosteric modulators of the α7 nAChR. Biological evaluation showed that phloretin, demethoxycurcumin, and bis-demethoxicurcuming behave as PAMs of α7 nAChR. In addition, some new curcumin derivatives were able to enhance the signal evoked by ACh; the activity values found for the tetrahydrocurcuminoid analog 23 were especially promising.


Subject(s)
Diarylheptanoids/pharmacology , Flavonoids/pharmacology , alpha7 Nicotinic Acetylcholine Receptor/agonists , Allosteric Regulation/drug effects , Animals , Biological Products/pharmacology , Curcumin/analogs & derivatives , Curcumin/chemical synthesis , Curcumin/pharmacology , Diarylheptanoids/chemistry , Dose-Response Relationship, Drug , Electrophysiological Phenomena/drug effects , Evoked Potentials/drug effects , Female , Xenopus laevis
3.
ACS Chem Neurosci ; 10(8): 3900-3909, 2019 08 21.
Article in English | MEDLINE | ID: mdl-31322853

ABSTRACT

Acetylcholine α7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain as well as in neuropsychiatric, neurodegenerative, and inflammatory processes. Positive allosteric modulators (PAMs) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here, we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the α7 receptors and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in Alzheimer's disease (AD)-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aß1-42, with cell death almost completely prevented at 10 and 30 µM, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the complete Freund's adjuvant (CFA)-induced paw inflammation model after intraperitoneal administration.


Subject(s)
Allosteric Regulation/drug effects , Analgesics/pharmacology , Cell Survival/drug effects , Neurons/drug effects , Neuroprotective Agents/pharmacology , Pain/drug therapy , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Analgesics/therapeutic use , Animals , Cell Line, Tumor , Humans , Inflammation/metabolism , Neurons/metabolism , Pain/metabolism , Pain Measurement , Rats
4.
Dev Comp Immunol ; 84: 279-291, 2018 07.
Article in English | MEDLINE | ID: mdl-29501534

ABSTRACT

The inflammatory reflex modulates the innate immune system, keeping in check the detrimental consequences of overstimulation. A key player controlling the inflammatory reflex is the alpha 7 acetylcholine receptor (α7nAChR). This receptor is one of the signalling molecules regulating cytokine expression in macrophages. In this study, we characterize a novel teleost α7nAChR. Protein sequence analysis shows a high degree of conservation with mammalian orthologs and trout α7nAChR has all the features and essential amino acids to form a fully functional receptor. We demonstrate that trout macrophages can bind α-bungarotoxin (α-BTX), a competitive antagonist for α7nAChRs. Moreover, nicotine stimulation produces a decrease in pro-inflammatory cytokine expression after stimulation with poly(I:C). These results suggest the presence of a functional α7nAChR in the macrophage plasma membrane. Further, in vivo injection of poly(I:C) induced an increase in serum ACh levels in rainbow trout. Our results manifest for the first time the functional conservation of the inflammatory reflex in teleosts.


Subject(s)
Fish Proteins/metabolism , Inflammation/immunology , Macrophages/immunology , Trout/physiology , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Animals , Biological Evolution , Bungarotoxins/pharmacology , Cells, Cultured , Conserved Sequence/genetics , Fish Proteins/genetics , Mammals , Nicotine/metabolism , Poly I-C/immunology , Reflex , Signal Transduction , alpha7 Nicotinic Acetylcholine Receptor/antagonists & inhibitors , alpha7 Nicotinic Acetylcholine Receptor/genetics
5.
Pflugers Arch ; 470(1): 13-20, 2018 01.
Article in English | MEDLINE | ID: mdl-28791474

ABSTRACT

In the adrenal gland, acetylcholine released on stimulation of the sympathetic splanchnic nerve activates neuronal-type nicotinic receptors (nAChRs) in chromaffin cells and triggers catecholamine secretion. At least two subtypes of nAChRs have been described in bovine chromaffin cells. The main subtype, a heteromeric assembly of α3, ß4 and perhaps α5 subunits, is involved in the activation step of the catecholamine secretion process and is not blocked by the snake toxin α-bungarotoxin. The other is α-bungarotoxin-sensitive, and its functional role has not yet been well defined. The α7 subunit conforms the homomeric structure of this subtype. All nAChR subunits share the same molecular organization and structural data at atomic resolution level are now available for some homomeric and heteromeric ensembles. The α3, ß4 and α5 subunits are clustered in genomes of different species, with the transcription factor Sp1 playing a co-ordinating role in the transcriptional regulation of these three subunits. The transcription factor Egr-1 controls the differential expression of α7 nAChR in adrenergic chromaffin cells, as happens with the enzyme phenylethanolamine N-methyl transferase. For unknown reasons, whole cell currents observed in bovine chromaffin cells clearly differ of the ones observed when different combinations of subunit RNAs are injected in oocytes. In addition to the typical nicotinic ligands, a variety of unrelated substances with clinical relevance can target nAChRs in chromaffin cells and, therefore, affect catecholamine secretion. They can act as agonists, antagonists or allosteric modulators.


Subject(s)
Chromaffin Cells/metabolism , Receptors, Nicotinic/metabolism , Animals , Chromaffin Cells/drug effects , Humans , Nicotinic Agonists/pharmacology , Nicotinic Antagonists/pharmacology , Protein Multimerization , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics
6.
Eur J Med Chem ; 143: 157-165, 2018 Jan 01.
Article in English | MEDLINE | ID: mdl-29174812

ABSTRACT

α7 Nicotinic acetylcholine receptors (nAChRs) are ion channels implicated in a number of CNS pathological processes, including pain and psychiatric, cognitive and inflammatory diseases. Comparing with orthosteric agonism, positive allosteric modulation of these channels constitutes an interesting approach to achieve selectivity versus other nicotinic receptors. We have recently described new chalcones and 1,3-diphenylpropanones as positive allosteric modulators (PAMs) of α7 nAChRs, which proved to have good analgesic activities but poor pharmacokinetic properties. Here we report the preparation of amino acid and peptide derivatives as prodrugs of these modulators with the aim of improving their in vivo biological activity. While the valine derivative showed very short half life in aqueous solutions to be considered a prodrug, Val-Val and Val-Pro-Val are suitable precursors of the parent 1,3-diphenylpropanones, via chemical and enzymatic transformation, respectively. Compounds 19 (Val-Val) and 21 (Val-Pro-Val), prodrugs of the 2',5',4-trihydroxy-1,3-diphenylpropan-1-one 3, showed significant antinociceptive activity in in vivo assays. The best compound, 21, displayed a better profile in the analgesia test than its parent compound 3, exhibiting about the same potency but long-lasting effects.


Subject(s)
Amino Acids/pharmacology , Analgesics/pharmacology , Pain/drug therapy , Peptides/pharmacology , Phenylpropionates/pharmacology , Prodrugs/pharmacology , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Allosteric Regulation/drug effects , Amino Acids/chemical synthesis , Amino Acids/chemistry , Analgesics/chemical synthesis , Analgesics/chemistry , Animals , Dose-Response Relationship, Drug , Edema/chemically induced , Edema/drug therapy , Freund's Adjuvant , Humans , Inflammation/chemically induced , Inflammation/drug therapy , Male , Molecular Structure , Pain Measurement , Peptides/chemical synthesis , Peptides/chemistry , Phenylpropionates/chemical synthesis , Phenylpropionates/chemistry , Prodrugs/chemical synthesis , Prodrugs/chemistry , Rats , Rats, Wistar , Structure-Activity Relationship , Xenopus
7.
ACS Chem Neurosci ; 7(8): 1157-65, 2016 08 17.
Article in English | MEDLINE | ID: mdl-27254782

ABSTRACT

A series of multitarget directed propargylamines, as well as other differently susbstituted piperidines have been screened as potential modulators of neuronal nicotinic acetylcholine receptors (nAChRs). Most of them showed antagonist actions on α7 nAChRs. Especially, compounds 13, 26, and 38 displayed submicromolar IC50 values on homomeric α7 nAChRs, whereas they were less effective on heteromeric α3ß4 and α4ß2 nAChRs (up to 20-fold higher IC50 values in the case of 13). Antagonism was concentration dependent and noncompetitive, suggesting that these compounds behave as negative allosteric modulators of nAChRs. Upon the study of a series of less complex derivatives, the N-benzylpiperidine motif, common to these compounds, was found to be the main pharmacophoric group. Thus, 2-(1-benzylpiperidin-4-yl)-ethylamine (48) showed an inhibitory potency comparable to the one of the previous compounds and also a clear preference for α7 nAChRs. In a neuroblastoma cell line, representative compounds 13 and 48 also inhibited, in a concentration-dependent manner, cytosolic Ca(2+) signals mediated by nAChRs. Finally, compounds 38 and 13 inhibited 5-HT3A serotonin receptors whereas they had no effect on α1 glycine receptors. Given the multifactorial nature of many pathologies in which nAChRs are involved, these piperidine antagonists could have a therapeutic potential in cases where cholinergic activity has to be negatively modulated.


Subject(s)
Membrane Potentials/drug effects , Nicotinic Antagonists/pharmacology , Piperidines/chemistry , Piperidines/pharmacology , alpha7 Nicotinic Acetylcholine Receptor/antagonists & inhibitors , Acetylcholine/pharmacology , Animals , Calcium/metabolism , Cell Line, Tumor , Dose-Response Relationship, Drug , Inhibitory Concentration 50 , Isoxazoles/pharmacology , Membrane Potentials/genetics , Microinjections , Neuroblastoma/pathology , Nicotinic Agonists/pharmacology , Oocytes , Patch-Clamp Techniques , Phenylurea Compounds/pharmacology , Receptors, Nicotinic , Receptors, Serotonin, 5-HT3/metabolism , Serotonin/pharmacology , Xenopus laevis , alpha7 Nicotinic Acetylcholine Receptor/genetics , alpha7 Nicotinic Acetylcholine Receptor/metabolism
8.
Future Med Chem ; 8(7): 731-49, 2016 05.
Article in English | MEDLINE | ID: mdl-27161515

ABSTRACT

UNLABELLED: Nicotine acethylcholine receptors (nAChRs) play critical roles in cognitive processes, neuroprotection and inflammation. RESULTS: According to their substituents, 1,3-diphenylpropan-1-one derivatives act as α7 nAChRs negative allosteric modulators (NAM, OMe) or Type I positive allosteric modulators (PAMs, OH). Compounds 7 and 31 were the most effective (989 and 666% enhancement of ACh-induced currents) and potent (EC50: 12.9 and 6.85 µM) PAMs. They exhibited strong radical scavenging values. Compound 31, selective over other neuronal nAChR subtypes and with acceptable pharmacokinetic profile, showed antinociceptive effects in a model of inflammatory pain. CONCLUSION: Compound 31 is a novel, potent and selective α7 nAChR PAM, displaying antioxidant and analgesic activities. The 1,3-diphenylpropan-1-one scaffold could be the base toward more advanced type I PAMs for the treatment of nAChR-mediated diseases.


Subject(s)
Analgesics/pharmacology , Antioxidants/pharmacology , Propane/analogs & derivatives , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Allosteric Regulation , Analgesics/chemistry , Animals , Antioxidants/chemistry , Gene Expression , Humans , Propane/chemistry , Propane/pharmacology , Rats, Wistar , Structure-Activity Relationship , alpha7 Nicotinic Acetylcholine Receptor/chemistry
9.
Eur J Med Chem ; 86: 724-39, 2014 Oct 30.
Article in English | MEDLINE | ID: mdl-25232969

ABSTRACT

The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α7 nicotinic function. The most potent compound (2,4,2',5'-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure.


Subject(s)
Analgesics/pharmacology , Chalcones/pharmacology , alpha7 Nicotinic Acetylcholine Receptor/antagonists & inhibitors , Allosteric Regulation/drug effects , Analgesics/chemical synthesis , Analgesics/chemistry , Animals , Behavior, Animal/drug effects , Cell Death/drug effects , Cell Survival/drug effects , Chalcones/chemical synthesis , Chalcones/chemistry , Dose-Response Relationship, Drug , Humans , Male , Maze Learning/drug effects , Mice , Molecular Structure , Oligomycins/antagonists & inhibitors , Oligomycins/pharmacology , Pain/drug therapy , Rats , Rats, Wistar , Rotenone/antagonists & inhibitors , Rotenone/pharmacology , Structure-Activity Relationship , alpha7 Nicotinic Acetylcholine Receptor/metabolism
10.
ACS Chem Neurosci ; 5(8): 683-9, 2014 Aug 20.
Article in English | MEDLINE | ID: mdl-24999981

ABSTRACT

We have characterized the effect of triazine derivatives on neuronal nicotinic receptors expressed in Xenopus oocytes. All triazines investigated inhibit the current of α7 and α3ß4 neuronal nicotinic receptors elicited by acetylcholine. The effect is concentration dependent, reversible, and noncompetitive. In contrast, some derivatives have a dual effect on α4ß2 receptors, by potentiating the currents at intermediate concentration and causing inhibition at higher concentrations. Triazine derivatives also affect the macroscopic kinetics of the heteromeric receptors α3ß4 and α4ß2 accelerating the rise and decay time course of the currents, but have no significant effect on the kinetics of homomeric α7 receptors. Two simple kinetic models are presented. The first reproduces the effects of different concentrations of triazines both on the peak currents and on the macroscopic kinetics of α7 with a simple inhibitory result. The second model describes the behavior of α4ß2 receptors involving a more complex dual action.


Subject(s)
Nicotinic Antagonists/pharmacology , Receptors, Nicotinic/metabolism , Triazines/pharmacology , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Animals , Dose-Response Relationship, Drug , Kinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Models, Neurological , Molecular Structure , Nicotinic Agonists/chemistry , Nicotinic Agonists/pharmacology , Nicotinic Antagonists/chemistry , Oocytes , Patch-Clamp Techniques , Receptors, Glycine/metabolism , Receptors, Serotonin/metabolism , Serotonin Antagonists/chemistry , Serotonin Antagonists/pharmacology , Triazines/chemistry , Xenopus laevis
11.
J Neurochem ; 123(4): 504-14, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22913551

ABSTRACT

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and ß2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or ß4 subunits reduced to about half, surface α-bungarotoxin binding sites and acetylcholine-gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only ß4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, ß4, α5, and α7 mRNAs, finding that α-bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3ß4(α5) receptors.


Subject(s)
Gene Expression Regulation/physiology , Protein Subunits/metabolism , Receptors, Nicotinic/metabolism , Acetylcholine/pharmacology , Analysis of Variance , Animals , Biophysics , Bungarotoxins/pharmacokinetics , Cattle , Cells, Cultured , Choline/pharmacology , Cholinergic Agents/pharmacology , Chromaffin Cells , Electric Stimulation , Gene Expression Regulation/genetics , Green Fluorescent Proteins/genetics , Humans , Iodine Isotopes/pharmacokinetics , Larva , Lipotropic Agents/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/genetics , Microinjections , Oocytes , Patch-Clamp Techniques , Protein Binding/drug effects , Protein Binding/physiology , Protein Subunits/genetics , RNA, Messenger/metabolism , Receptors, Nicotinic/genetics , Xenopus , alpha7 Nicotinic Acetylcholine Receptor
12.
FEBS Lett ; 585(15): 2477-80, 2011 Aug 04.
Article in English | MEDLINE | ID: mdl-21729697

ABSTRACT

Deletion of a small cytoplasmic fragment close to the fourth transmembrane segment of the nicotinic α7 receptor (Glu437 to Arg447) abolished membrane expression. Different single mutants showed moderate to strong decreases in expression whereas the latter was totally abolished upon proline substitutions. We hypothesize that preservation of an α-helix formed by the fourth transmembrane segment and the adjacent cytoplasmic region is essential for membrane receptor expression. Moreover, in selected mutants with low or null membrane expression, a significant proportion of mature receptors was present inside the cell. Hence, elements in this cytoplasmic fragment might influence receptor transport to the membrane.


Subject(s)
Mutation , Receptors, Nicotinic/analysis , Amino Acid Sequence , Amino Acid Substitution , Animals , Cattle , Cytoplasm/chemistry , Membrane Proteins/analysis , Protein Structure, Secondary , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics , alpha7 Nicotinic Acetylcholine Receptor
13.
J Neurochem ; 118(6): 968-78, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21740443

ABSTRACT

Activation of nicotinic acetylcholine receptors (nAChR) requires a global conformational change involving a number of domains of the protein. Structural data from Torpedo nAChR suggest that adjacent subunits might be functionally coupled at the interface between the ß-strand ß3 and the loop B through a salt bridge between α1Asp152 and γArg78. We have checked this hypothesis in homomeric α7 nAChRs by mutating residues at these (Gly152 and Arg79) and neighboring locations and analyzing the results obtained after expression of single and double mutants in Xenopus oocytes. We found that Arg79 mutants showed a decreased gating function when challenged with different agonists, being the reduction more important for dimethylphenylpiperazinium. EC(50) values in these mutants were also increased up to 30-fold. In contrast, mutating Gly152 only showed significant higher EC(50) values for ACh. However, all Gly153 mutants presented increased gating function and lower EC(50) values with no significant differences among them. When analyzing several mutant cycles it is concluded that Arg79 is functionally coupled to Gly152, but neither to Gly153 nor to Asp157. These data suggest an involvement of the minus side of homomeric α7 nAChRs in their gating function, reinforcing the significance of complementary subunits in the gating of neuronal nAChRs.


Subject(s)
Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Amino Acid Substitution , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cattle , DNA, Complementary/genetics , Data Interpretation, Statistical , Dimethylphenylpiperazinium Iodide/pharmacology , Electrophysiological Phenomena , Humans , Ion Channel Gating/drug effects , Ion Channel Gating/genetics , Models, Molecular , Mutation/genetics , Mutation/physiology , Nicotinic Agonists/pharmacology , Oocytes/drug effects , Pyridines/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Nicotinic/drug effects , Torpedo/genetics , Torpedo/metabolism , Xenopus laevis , alpha7 Nicotinic Acetylcholine Receptor
14.
J Neurochem ; 119(1): 40-9, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21790604

ABSTRACT

We have studied the role of different amino acids in the M2 transmembrane domain of the α7 neuronal nicotinic receptor by mutating residues that differ from the ones located at the same positions in other α (α2-α10) or ß (ß2-ß4) subunits. Our aim was to investigate the contribution of these amino acids to the peculiar kinetic and inward rectification properties that differentiate the homomeric α7 receptor from other nicotinic receptors. Mutations of several residues strongly modified receptor function. We found that Thr245 had the most profound effect when mutated to serine, an amino acid present in all heteromeric receptors composed of α and ß subunits, by dramatically increasing the maximal current, decreasing the decaying rate of the currents and decreasing receptor rectification. Some mutants also showed altered agonist-binding properties as revealed by shifts in the dose-response curves for acetylcholine. We conclude that residues in the M2 segment and flanking regions contribute to the unusual properties of the α7 receptor, especially to its characteristic fast kinetic behavior and strong inward rectification and furthermore to the potency of agonists.


Subject(s)
Ion Channel Gating/physiology , Receptors, Nicotinic/metabolism , Acetylcholine/metabolism , Algorithms , Amino Acid Sequence , Amino Acid Substitution , Animals , Bungarotoxins/metabolism , Cattle , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Electrophysiological Phenomena , Ion Channel Gating/genetics , Kinetics , Membrane Potentials/physiology , Models, Molecular , Molecular Sequence Data , Oocytes/drug effects , Oocytes/metabolism , Patch-Clamp Techniques , Protein Binding , Receptor, Muscarinic M2/drug effects , Receptor, Muscarinic M2/metabolism , Receptors, Nicotinic/genetics , Xenopus laevis , alpha7 Nicotinic Acetylcholine Receptor
15.
J Neurosci ; 30(30): 10112-26, 2010 Jul 28.
Article in English | MEDLINE | ID: mdl-20668195

ABSTRACT

The function of Ric-3, which is required for nicotinic acetylcholine receptor (nAChR) expression in C. elegans, is unclear. Here we found that Ric-3 can promote or inhibit cell-surface delivery of alpha-bungarotoxin-binding nAChRs (BgtRs) composed of alpha7 subunits. At low levels, Ric-3 promoted BgtR assembly, endoplasmic reticulum (ER) release, and cell-surface delivery without trafficking from the ER. At high Ric-3 levels, Ric-3 suppressed BgtR surface delivery, but not its assembly, and BgtRs were retained in the ER or in Ric-3-containing aggregates. In PC12 cells, native BgtRs trafficked to the cell surface from the ER where low levels of endogenous Ric-3 were observed. In cultured neurons, native Ric-3 levels were higher than in PC12 cells, and Ric-3 and alpha7 subunits were found in somata and dendrites, but not axons, of inhibitory interneurons. Ric-3 trafficked with alpha7 subunits in rapidly moving vesicles to dendrites, where it was restricted to the ER subcompartment. We conclude that Ric-3 has two potential functions. At low levels, Ric-3 interactions are short-lived and promote BgtR assembly and ER release. At higher levels, Ric-3 interactions are longer-lived and mediate ER retention. In neurons, Ric-3 ER retention appears to promote transport within the dendritic ER subcompartment, thereby restricting alpha7 trafficking to dendrites and preventing axonal transport.


Subject(s)
Dendrites/ultrastructure , Endoplasmic Reticulum/metabolism , Membrane Proteins/genetics , Molecular Chaperones/genetics , Acetylcholine/pharmacology , Animals , Autoantigens/metabolism , Bungarotoxins/metabolism , Bungarotoxins/pharmacology , Cell Line/cytology , Cells, Cultured , Chickens , Cholinergic Agents/pharmacology , Dendrites/drug effects , Endoplasmic Reticulum/drug effects , Flow Cytometry/methods , Glutamate Decarboxylase/metabolism , Green Fluorescent Proteins/genetics , Hippocampus/cytology , Humans , Iodine Isotopes/metabolism , Membrane Potentials/drug effects , Membrane Potentials/genetics , Membrane Proteins/metabolism , Mice , Models, Biological , Patch-Clamp Techniques/methods , Protein Binding/drug effects , Protein Binding/genetics , Protein Disulfide-Isomerases/metabolism , Protein Transport/genetics , Rats , Receptors, Nicotinic/metabolism , Tissue Distribution/drug effects , Transfection/methods , alpha7 Nicotinic Acetylcholine Receptor
16.
J Neurochem ; 113(4): 1036-45, 2010 May.
Article in English | MEDLINE | ID: mdl-20420581

ABSTRACT

Nicotinic acetylcholine receptors (nAChRs) transmit the agonist signal to the channel gate through a number of extracellular domains. We have previously shown that particular details of the process of coupling binding to gating could be quantitative and qualitatively different in muscle and neuronal type nAChRs. We have extended previous studies on homomeric alpha7 nAChRs to heteromeric alpha3beta4 nAChRs, by mutating residues located at loops 2 and 7, and M2-M3 linker of both alpha3 and beta4 subunits which, in order to monitor surface expression, were modified to bind alpha-bungarotoxin, and expressed in Xenopus oocytes. We show that, in general, mutations in these domains of both alpha3 and beta4 subunits affect the gating function, although the effects are slightly larger if they are inserted in the alpha3 subunit. However, the involvement of a previously reported intrasubunit interaction in coupling (Gln48-Ile130) seems to be restricted to the beta4 subunit. We also show that mutations at these domains, particularly loop 2 of the alpha3 subunit, change the pharmacological profile of alpha3beta4 nAChRs, decreasing nicotine's and increasing cytisine's effectiveness relative to acetylcholine. It is concluded that, unlike muscle nAChRs, the non-alpha subunits play a relevant role in the coupling process of neuronal alpha3beta4 nAChRs.


Subject(s)
Cell Membrane/chemistry , Ion Channel Gating/genetics , Receptors, Nicotinic/chemistry , Acetylcholine/metabolism , Acetylcholine/pharmacology , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Female , Humans , Ion Channel Gating/drug effects , Mutation/genetics , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/genetics , Oocytes , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Protein Subunits/chemistry , Protein Subunits/drug effects , Protein Subunits/genetics , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/genetics , Synaptic Transmission/drug effects , Synaptic Transmission/genetics , Xenopus laevis
17.
Biochim Biophys Acta ; 1798(3): 654-9, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20043866

ABSTRACT

We have studied the role of loop 9 in the function of neuronal nicotinic receptors. By systematically mutating the residues in the loop we have determined that the most important amino acids determining the coupling of binding to gating are the ones closer to the transmembrane region. Single mutations at location E173 in homomeric alpha7 receptors destroyed their function by completely abolishing the current while preserving the expression at the membrane. In contrast, heteromeric receptor alpha3beta4 with the same mutations retained some function. We conclude that loop 9 has a different role in the function of homomeric and heteromeric receptors.


Subject(s)
Neurons/metabolism , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/metabolism , Acetylcholine/pharmacology , Alanine/genetics , Animals , Cattle , Humans , Ion Channel Gating/drug effects , Mutagenesis/drug effects , Mutant Proteins/metabolism , Mutation/genetics , Protein Multimerization , Protein Structure, Secondary , Rats , Structure-Activity Relationship , Xenopus laevis
18.
J Neurochem ; 112(1): 103-11, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19840217

ABSTRACT

Recently, we have shown that the alpha-helix present at the N-termini of alpha7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking beta-strands beta2 and beta3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant alpha7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (alpha3beta4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric alpha7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the alpha-helix is an important requirement for receptor assembly.


Subject(s)
Protein Subunits/biosynthesis , Protein Subunits/genetics , Receptors, Nicotinic/biosynthesis , Receptors, Nicotinic/genetics , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , Cattle , Female , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Chimeric Proteins/biosynthesis , Mutant Chimeric Proteins/genetics , Protein Binding/genetics , Protein Structure, Secondary/genetics , Protein Subunits/metabolism , Receptors, Nicotinic/metabolism , Xenopus laevis , alpha7 Nicotinic Acetylcholine Receptor
19.
FEBS Lett ; 583(6): 1045-51, 2009 Mar 18.
Article in English | MEDLINE | ID: mdl-19236866

ABSTRACT

We have studied the role of the highly conserved residue alphaLysine145 in the early steps of activation by acetylcholine of the nicotinic acetylcholine receptor (nAChR). Both macroscopic and single-channel currents were recorded in the slowly desensitizing chimeric mutant receptor alpha7V201-5HT3A/R432Q/R436D/R440A, made of alpha7 nAChRs and serotonin receptors of subtype 3A (ch1), and its corresponding mutant K145A (ch1/K145A) expressed in Xenopus oocytes. Mutant ch1/K145A receptors had a reduced gating function similar to that produced by the same mutation in the wild type receptor alpha7. The mutated receptor has reduced opening rate constants, beta, and increased closing rate constants, alpha.


Subject(s)
Ion Channel Gating/physiology , Receptors, Nicotinic/metabolism , Receptors, Serotonin, 5-HT3/metabolism , Recombinant Fusion Proteins/metabolism , Alkaloids/pharmacology , Animals , Azocines/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Carbachol/pharmacology , Cattle , Cholinergic Agonists/pharmacology , Ion Channel Gating/drug effects , Models, Biological , Nicotine/pharmacology , Patch-Clamp Techniques , Protein Binding , Pyridines/pharmacology , Quinolizines/pharmacology , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/physiology , Recombinant Fusion Proteins/agonists , Recombinant Fusion Proteins/physiology , Serotonin 5-HT3 Receptor Agonists , Xenopus , alpha7 Nicotinic Acetylcholine Receptor
20.
J Neurochem ; 108(6): 1399-409, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19166504

ABSTRACT

We studied the role of the alpha-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in alpha7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the alpha-helix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of alpha7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (alpha3beta4 and alpha4beta2) and 5-HT(3A) receptors also abolished their expression at the membrane. We conclude that the N-terminal alpha-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.


Subject(s)
Receptors, Nicotinic/chemistry , Receptors, Nicotinic/physiology , Animals , Bungarotoxins/metabolism , Cattle , Cell Line, Tumor , Green Fluorescent Proteins/genetics , Leucine/genetics , Models, Molecular , Mutagenesis/physiology , Mutation/genetics , Neuroblastoma , Oocytes , Proline/genetics , Protein Structure, Secondary/genetics , Protein Structure, Secondary/physiology , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/genetics , Receptors, Serotonin, 5-HT3/genetics , Transfection/methods , Xenopus , alpha7 Nicotinic Acetylcholine Receptor
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