5-I A-85380 and TC-2559 differentially activate heterologously expressed α4β2 nicotinic receptors (original) (raw)
Related papers
5-I A-85380 and TC-2559 differentially activate heterologously expressed a4�2 nicotinic receptors
Eur J Pharmacol, 2006
The neuronal nicotinic acetylcholine receptor α4 and β2 subunits expressed in heterologous expression systems assemble into at least two distinct subunit stoichiometries of α4β2 receptor. The (α4)2(β2)3 stoichiometry is about 100-fold more sensitive to acetylcholine than the (α4)3(β2)2 stoichiometry. In order to investigate if agonists in general distinguish high- and low-affinity α4β2 nicotinic acetylcholine receptors, we have expressed human α4 and β2 nicotinic acetylcholine receptor subunits in two different expression systems. The relative amounts of α4β2 nicotinic acetylcholine receptors with high- and low-affinity for acetylcholine were manipulated by (a) injecting the subunit cDNAs at different α:β ratios into Xenopus oocytes and (b) by culturing HEK-293 cells stably expressing α4β2 nicotinic acetylcholine receptors overnight at different temperatures. The sensitivities of the α4β2 nicotinic acetylcholine receptors to the agonists acetylcholine, 5-I A-85380, and TC-2559 were investigated using the voltage-clamp technique on Xenopus oocytes and using a fluorescent imaging plate reader to measure calcium responses from HEK-293 cells. Like acetylcholine, 5-I A-85380 produced biphasic concentration–response curves and the high-affinity component became larger when the cells were manipulated to produce a greater proportion of (α4)2(β2)3 nicotinic acetylcholine receptors. Interestingly, under all circumstances, TC-2559 produced monophasic concentration–response curves. In oocytes injected with α4 and β2 subunits in the 1:1 ratio the maximum effect of TC-2559 was 28% of that of acetylcholine. The EC50 for TC-2559 was not changed when oocytes were manipulated to express exclusively (α4)2(β2)3 nicotinic acetylcholine receptors, however, the maximum effect of TC-2559 was dramatically enhanced. These results suggest that TC-2559 is a selective agonist of the (α4)2(β2)3 nicotinic acetylcholine receptor stoichiometry.
Stable expression and pharmacological properties of the human α 7 nicotinic acetylcholine receptor
European Journal of Pharmacology: Molecular Pharmacology, 1995
The a 7 neuronal nicotinic acetylcholine receptor subtype forms a Ca-'--permeable homooligomeric ion channel sens a-bungarotoxin in Xenopus oocytes. In this study, we have stably and functionally expressed the human a 7 eDNA in a mammal line, HEK-293 and examined its pharmacologic properties. ['2~l]a-Bungarotoxin bound to transfected cells with a K d value of and a B,.,~,, value of 973 pmol/mg protein. No specific binding was detected in untransfected cells. Specific binding could be di by unlabeled a-bungarotoxin (g i = 0.5 riM) and an excellent correlation was observed between binding affinities of a series of n cholinergic ligands in transfected cells and those in the human neuroblastoma IMR-32 cell line. Additionally, cell surface expre~ a 7 receptors was detected by fluorescein isothiocyanate-conjugated a-bungarotoxin in transfected cells. Whole cell currents sen., blockade by a-bungarotoxin, and with fast kinetics of activation and inactivation, were recorded from transfected cells upo application of (-)-nicotine or acetylcholine with E(75o values of 49 jzM and 155 /.,tM respectively. We conclude that the hut subunit when expressed alone can form functional ion channels and that the stably transfected HEK-293 cell line serves as a system for studying human a 7 nicotinic receptor function and regulation, and for examining ligand interactions.
Function of Mammalian Nicotinic Acetylcholine Receptors
Nicotinic Acetylcholine Receptor, 1986
This paper will briefly review the results of our studies of the function of the nicotinic acetylcholine receptors (AChRs) expressed by clonal mammalian BC3H-1 cells in vitro. The observations and interpretations will be compared with expectations based on the physiology of junctional transmission and with data obtained by others. BC3H-1 cells are a cell line which originated in a brain tumor of C3H mice (Schubert et al., 1974). These cells differentiate in culture and express a large number of AChR. A number of studies have established that the AChR are similar to skeletal muscle receptors biochemically (Boulter and Patrick, 1977), pharmacologically (Patrick et al., 1977; Sine and Taylor, 1981) and in the primary amino acid sequence of the subunits (LaPolla et al., 1984; Boulter et aI, 1985). The methods used have been described (Sine and Steinbach, 1984a,b; 1986a,b,c). The data discussed here were obtained at 11°C using cell attached patches. Cells
FEBS letters, 1996
The tz31]~4 subtype of neuronal nicotinic acetylcholine receptor (nAChR) was stably expressed in human embryonic kidney (HEK) 293 cells that co-expressed a voltage-gated Ca 2+ channel. ¢z3/134-nAChR-expressing clones were identified using the fura-2 Ca 2÷ imaging technique, and were further characterised by single-cell and whole-cell patch-clamp studies. Acetylcholine (ACh) induced fast activating currents which showed desensitisation and inward rectification. The conductance of the ACh-activated channel was 29 pS. The order of potency of the nicotinic agonists tested was cytisine -=-nicotine > acetylcholine. The ECs0 value for ACh was 145 I~VI; the Hill coefficient was close to 2. The currents elicited by ACh were effectively blocked by nicotinic antagonists, but not by the muscarinic antagonist atropine. These properties are comparable to the pharmacological and physiological profile of ganglionic nicotinic receptors and type III currents of cultured Idppocampal neurons.
Nicotinic acetylcholine receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database
IUPHAR/BPS guide to pharmacology CITE, 2019
Nicotinic acetylcholine receptors are members of the Cys-loop family of transmitter-gated ion channels that includes the GABA A , strychnine-sensitive glycine and 5-HT 3 receptors [210, 3, 155, 220, 252]. All nicotinic receptors are pentamers in which each of the five subunits contains four α-helical transmembrane domains. Genes encoding a total of 17 subunits (α1-10, β1-4, γ, δ and ε) have been identified [117]. All subunits with the exception of α8 (present in avian species) have been identified in mammals. All α subunits possess two tandem cysteine residues near to the site involved in acetylcholine binding, and subunits not named α lack these residues [155]. The orthosteric ligand binding site is formed by residues within at least three peptide domains on the α subunit (principal component), and three on the adjacent subunit (complementary component). nAChRs contain several allosteric modulatory sites. One such site, for positive allosteric modulators (PAMs) and allosteric agonists, has been proposed to reside within an intrasubunit cavity between the four transmembrane domains [257, 85]; see also [103]). The high resolution crystal structure of the molluscan acetylcholine binding protein, a structural homologue of the extracellular binding domain of a nicotinic receptor pentamer, in complex with several nicotinic receptor ligands (e.g.[33]) and the crystal structure of the extracellular domain of the α1 subunit bound to α-bungarotoxin at 1.94 Å resolution [ 53], has revealed the orthosteric binding site in detail (reviewed in [210, 117, 37, 193]). Nicotinic receptors at the somatic neuromuscular junction of adult animals have the stoichiometry (α1) 2 β1δε, whereas an extrajunctional (α1) 2 β1γδ receptor predominates in embryonic and denervated skeletal muscle and other pathological states. Other nicotinic receptors are assembled as combinations of α(2-6) and &beta(2-4) subunits. For α2, α3, α4 and β2 and β4 subunits, pairwise combinations of α and β (e.g. α3β4 and α4β2) are sufficient to form a functional receptor in vitro, but far more complex isoforms may exist in vivo (reviewed in [94, 91, 155]). There is strong evidence that the pairwise assembly of some α and β subunits can occur with variable stoichiometry [e.g. (α4) 2 (β2) 2 or (α4) 3 (β2) 2 ] which influences the biophysical and pharmacological properties of the receptor [155]. α5 and β3 subunits lack function when expressed alone, or pairwise, but participate in the formation of functional hetero-oligomeric receptors when expressed as a third subunit with another α and β pair [e.g. α4α5αβ2, α4αβ2β3, α5α6β2, see [155] for further examples]. The α6 subunit can form a functional receptor when co-expressed with β4 in vitro, but more efficient expression ensues from incorporation of a third partner, such as β3 [256]. The α7, α8, and α9 subunits form functional homo-oligomers, but can also combine with a second subunit to constitute a hetero-oligomeric assembly (e.g. α7β2 and α9α10). For functional expression of the α10 subunit, co-assembly with α9 is necessary. The latter, along with the α10 subunit, appears to be largely confined to cochlear and vestibular hair cells. Comprehensive listings of nicotinic receptor subunit combinations identified from recombinant expression systems, or in vivo, are given in [ 155]. In addition, numerous proteins interact with nicotinic ACh receptors modifying their assembly, trafficking to and from the cell surface, and activation by ACh (reviewed by [154, 9, 115]). The nicotinic receptor Subcommittee of NC-IUPHAR has recommended a nomenclature and classification scheme for nicotinic acetylcholine (nACh) receptors based on the subunit composition of known, naturallyand/or heterologously-expressed nACh receptor subtypes [139]. Headings for this table reflect abbreviations designating nACh receptor subtypes based on the predominant α subunit contained in that receptor subtype. An asterisk following the indicated α subunit denotes that other subunits are known to, or may, assemble with the indicated α subunit to form the designated nACh receptor subtype(s). Where subunit stoichiometries within a specific nACh receptor subtype are known, numbers of a particular subunit larger than 1 are indicated by a subscript following the subunit (enclosed in parentheses see also [44]). Contents This is a citation summary for Nicotinic acetylcholine receptors in the Guide to Pharmacology database (GtoPdb). It exists purely as an adjunct to the database to facilitate the recognition of citations to and from the database by citation analyzers. Readers will almost certainly want to visit the relevant sections of the database which are given here under database links. GtoPdb is an expert-driven guide to pharmacological targets and the substances that act on them. GtoPdb is a reference work which is most usefully represented as an on-line database. As in any publication this work should be appropriately cited, and the papers it cites should also be recognized. This document provides a citation for the relevant parts of the database, and also provides a reference list for the research cited by those parts. Please note that the database version for the citations given in GtoPdb are to the most recent preceding version in which the family or its subfamilies and targets were substantially changed. The links below are to the current version. If you need to consult the cited version, rather than the most recent version, please contact the GtoPdb curators.
Nicotinic Acetylcholine Receptors Assembled from the alpha 7 and beta 3 Subunits
Journal of Biological Chemistry, 1999
Intracellular recordings were performed in voltageclamped Xenopus oocytes upon injection with a mixture of cDNAs encoding the 3 and mutant ␣7 ( L247T ␣7) neuronal nicotinic acetylcholine receptor (nAChR) subunits. The expressed receptors maintained sensitivity to methyllycaconitine and to ␣-bungarotoxin but exhibited a functional profile strikingly different from that of the homomeric L247T ␣7 receptor. The heteromeric L247T ␣73 nAChR had a lower apparent affinity and a faster rate of desensitization than L247T ␣7 nAChR, exhibited nonlinearity in the I-V relationship, and was inhibited by 5-hydroxytryptamine, much like wild type ␣7 ( WT ␣7) nAChR. Single channel recordings in cell-attached mode revealed unitary events with a slope conductance of 19 picosiemens and a lifetime of 5 ms, both values being much smaller than those of the homomeric receptor channel. Upon injection with a mixture of WT ␣7 and 3 cDNAs, clear evidence was obtained for the plasma membrane assembly of heteromeric nAChRs, although ACh could not activate these receptors. It is concluded that 3, long believed to be an orphan subunit, readily co-assembles with other subunits to form heteromeric receptors, some of which may be negative regulators of cholinergic function.
Molecular pharmacology, 2017
The α4β2 nicotinic acetylcholine receptor (nAChR) is important in central nervous system physiology and in mediating several of the pharmacological effects of nicotine on cognition, attention, and affective states. It is also the likely receptor that mediates nicotine addiction. This receptor assembles in two distinct stoichiometries: (α4)2(β2)3 and (α4)3(β2)2, which are referred to as high-sensitivity (HS) and low-sensitivity (LS) nAChRs, respectively, based on a difference in the potency of acetylcholine to activate them. The physiologic and pharmacological differences between these two receptor subtypes have been described in heterologous expression systems. However, the presence of each stoichiometry in native tissue currently remains unknown. In this study, different ratios of rat α4 and β2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS α4β2 nAChRs expressed in a mammalian cell line. The HS and LS nAChRs were char...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1998
To identify the molecular determinants underlying the pharmacological diversity of neuronal nicotinic acetylcholine receptors, we compared the alpha7 homo-oligomeric and alpha4beta2 hetero-oligomeric receptors. Sets of residues from the regions initially identified within the agonist binding site of the alpha4 subunit were introduced into the alpha7 agonist binding site, carried by the homo-oligomeric alpha7-V201-5HT3 chimera. Introduction of the alpha4 residues 183-191 into alpha7 subunit sequence (chimera C2) selectively increased the apparent affinities for equilibrium binding and for ion channel activation by acetylcholine, resulting in a receptor that no longer displays differences in the responses to acetylcholine and nicotine. Introduction of the alpha4 residues 151-155 (chimera B) produced a approximately 100-fold increase in the apparent affinity for both acetylcholine and nicotine in equilibrium binding measurements. In both cases electrophysiological recordings revealed a...
Molecular Pharmacology, 2006
␣4 and 2 nicotinic acetylcholine receptor (nAChR) subunits expressed heterologously assemble into receptors with high (HS) and low (LS) sensitivity to acetylcholine (ACh); their relative proportions depend on the ␣4 to 2 ratio. In this study, injection of oocytes with 1:10 ␣4/2 subunit cDNA ratios favored expression of HS ␣42 nAChRs, as evidenced by monophasic ACh concentration-response curves, whereas injections with 10:1 cDNA ratios favored expression of LS ␣42 receptors. The stoichiometry was inferred from the shifts in the ACh EC 50 values caused by Leu to Thr mutations at position 9Ј of the second transmembrane domain of ␣4 and 2. The 1:10 injection ratio produced the (␣4) 2 (2) 3 stoichiometry, whereas 10:1 injections produced the (␣4) 3 (2) 2 stoichiometry. The agonists epibatidine, 3-[2(S)-azetidinylmethoxy]pyridine (A-85380), 5-ethoxy-metanicotine (TC-2559), cytisine, and 3-Br-cytisine This work was supported by the Wellcome Trust (CRIG project) and by an Oxford Brookes University postgraduate scholarship (to M.M.).