The neuronal α6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells (original) (raw)
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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.
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.
2003
Fura-2 recording of Ca 2ϩ influx was used to show that incubation in 1 M nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHE-sensitive component that presumably corresponds to ␣42 receptors. To study changes in ␣42 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the ␣4 or 2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca 2ϩ influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent ␣4 and 2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of ␣42 receptors, coexpression of the ␣4-YFP and 2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic ␣42 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 M nicotine, there was increased FRET in the cell body, denoting increased assembly of ␣42 receptors. Thus, changes in ␣42 receptor assembly play a role in the regulation of ␣42 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca 2ϩ-stimulated pathways.
European Journal of Neuroscience, 1997
Within the chick central nervous system, expression of the 3 nicotinic acetylcholine receptor gene is restricted to a subset of retinal neurons, the majority of which are ganglion cells. Transient transfection in retinal neurons and in neural and non-neural cells from other regions of the chick embryo allowed the identification of the cis-regulatory domain of the 3 gene. Within this domain, a 75-base pair fragment located immediately upstream of the transcription start site suffices to reproduce the neuron-specific expression pattern of 3. This fragment encompasses an E-box and a CAAT box, both of which are shown to be key positive regulatory elements of the 3 promoter. Co-transfection experiments into retinal, telencephalic, and tectal neurons with plasmid reporters of 3 promoter activity and a number of vectors expressing different neuronal (ASH-1, NeuroM, NeuroD, CTF-4) and non-neuronal (MyoD) basic helix-loop-helix transcription factors indicate that the cis-regulatory domain of 3 has the remarkable property of discriminating accurately between related members of the basic helix-loop-helix protein family. The sequence located immediately 3 of the E-box participates in this selection, and the E-box acts in concert with the nearby CAAT box.
Molecular Pharmacology, 2007
Neuronal nicotinic acetylcholine (ACh) receptors are ligandgated, cation-selective ion channels. Nicotinic receptors containing ␣4, ␣6, 2, and 3 subunits are expressed in midbrain dopaminergic neurons, and they are implicated in the response to smoked nicotine. Here, we have studied the cell biological and biophysical properties of receptors containing ␣6 and 3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged 3 subunits were fully functional compared with receptors with untagged 3 subunits. We find that 3and ␣6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent ␣4 and 2 subunits in neuronal soma and dendrites. Fö rster resonance energy transfer (FRET) reveals efficient, specific assembly of 3 and ␣6 into nicotinic receptor pentamers of various subunit compositions.
Identification of nicotinic acetylcholine receptor subunit expression in early avian embryos
The FASEB Journal, 2013
In the CNS, receptor recycling is critical for synaptic plasticity; however, the recycling of receptors has never been observed at peripheral synapses. Using a novel imaging technique, we show here that nicotinic acetylcholine receptors (AChRs) recycle into the postsynaptic membrane of the neuromuscular junction. By sequentially labeling AChRs with biotin-bungarotoxin and streptavidin-fluorophore conjugates, we were able to distinguish recycled, preexisting, and new receptor pools at synapses in living mice. Time-lapse imaging revealed that recycled AChRs were incorporated into the synapse within hours of initial labeling, and their numbers increased with time. At fully functional synapses, AChR recycling was robust and comparable in magnitude with the insertion of newly synthesized receptors, whereas chronic synaptic activity blockade nearly abolished receptor recycling. Finally, using the same sequential labeling method, we found that acetylcholinesterase, another synaptic component, does not recycle. These results identify an activity-dependent AChRrecycling mechanism that enables the regulation of receptor density, which could lead to rapid alterations in synaptic efficacy.
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.