[31] Transient expression of heteromeric ion channels

doi:10.1016/S0076-6879(98)93034-8

Methods in Enzymology

Volume 293, 1998, Pages 564-585

https://doi.org/10.1016/S0076-6879(98)93034-8 Get rights and content

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A major advance in the study of ion channels has been the ability to express channels in foreign host cells after isolation of the channel genes. Initially, the system of choice for rapid expression of ion channel proteins was the Xenopus oocyte. Most of the electrophysiologic characterization of newly isolated ion channel subunit cDNAs has been performed using the Xenopus oocyte expression system. Unfortunately, the amount of ion channel protein produced using this system is relatively small. For biochemical and cell biological analysis, ion channels are generally expressed in cultured cell lines where production can be generated in enormous numbers of cells. The transfer of ion channel genes to cultured cells allows the synthesis of large amounts of these proteins, which previously could only be purified in minute quantities after much effort. Cultured cells have the additional advantage over Xenopus oocytes in providing an environment that is usually closer to that of the expressed ion channel's native environment.This chapter presents a step-by-step description of the methods for ion channel gene transfer into cultured cell lines. The intent here is to describe problems and complexities that have been encountered using these methods and to discuss changes that have been made in the methods to counter problems and to achieve higher levels of expression.

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Cited by (22)

Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes
2016, Biophysical Journal
Citation Excerpt :
Thus, we hypothesize that the dominant negative functional effects observed by the overexpression of LRRC8A may be caused by the formation of nonfunctional stoichiometries. Similar dominant negative effects are observed when overexpressing nicotinic receptors (44), probably by having complexes of nonnative stoichiometries. Thus, we envisage that changing the relative proportion of each subunit may be a way to regulate VRAC channel activity.
Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.
High affinity binding of epibatidine to serotonin type 3 receptors
2008, Journal of Biological Chemistry
Citation Excerpt :
Hemagglutinin epitope-tagged mouse 5HT3A was subcloned into the pcDNA3.1 vector using standard methodology. HEK cells were transiently transfected with the subunit cDNA constructs using a calcium phosphate protocol (53). In some experiments, for comparison with mouse 5-HT3A receptors, HEK cells were transfected with cDNA encoding the human 5-HT3A receptor subunit.
Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of ¹²⁵I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT₃Rs) block the remaining 25%. ¹²⁵I-Epibatidine binds with a high affinity to native 5-HT₃Rs of N1E-115 cells and to receptors composed of only 5-HT_3A subunits expressed in HEK cells. In these cells, serotonin, the 5-HT₃R-specific antagonist MDL72222, and the 5-HT₃R agonist chlorophenylbiguanide readily competed with ¹²⁵I-epibatidine binding to 5-HT₃Rs. Nicotine was a poor competitor for ¹²⁵I-epibatidine binding to 5-HT₃Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of ¹²⁵I-epibatidine binding to 5-HT₃Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT₃Rs in neuroblastoma cell lines and 5-HT_3ARs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT₃Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT₃R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT₃Rs.
Up-regulation of nicotinic receptors by nicotine varies with receptor subtype
2008, Journal of Biological Chemistry
Citation Excerpt :
Unless otherwise noted, cells were moved to 30 °C 24 h before experimentation. DNA was expressed by transient transfection into tsA201 cells using the calcium phosphate method (31). Stable cell lines were established from tsA201 cells using calcium phosphate transfection and hygromycin B selection (Calbiochem) at 0.42 mg/ml.
Recent evidence suggests that in addition to α4β2 and α3-containing nicotinic receptors, α6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that α6β2, like α3β2 and α4β2 receptors, formed high affinity epibatidine binding complexes that are pentameric, trafficked to the cell surface, and produced acetylcholine-evoked currents. Chronic nicotine exposure up-regulated α6β2 receptors with differences in up-regulation time course and concentration dependence compared with α4β2 receptors, the predominant high affinity nicotine binding site in brain. The α6β2 receptor up-regulation required higher nicotine concentrations than for α4β2 but lower than for α3β2 receptors. The α6β2 up-regulation occurred 10-fold faster than for α4β2 and slightly faster than for α3β2. Our data suggest that nicotinic receptor up-regulation is subtype-specific such that α6-containing receptors up-regulate in response to transient, high nicotine exposures, whereas sustained, low nicotine exposures up-regulate α4β2 receptors.
Endoplasmic reticulum chaperones stabilize nicotinic receptor subunits and regulate receptor assembly
2007, Journal of Biological Chemistry
Citation Excerpt :
The CN-specific primers used are a forward primer (5′-TAC AAG GAC GAT GAT GAC AAG CAA GAG GAG GAC GAT AGG AAA CC-3′) and a reverse primer (5′-GTC ATC ATC GTC CTT GTA GTC ACT GGC AGT GCC GCC ATC TTC TTC AGC-3′), where the underlined base pairs are the FLAG epitope tag. Transient Transfection—The chaperones, Torpedo and mouse AChR subunit cDNAs, were transiently transfected into 6-cm cultures of tsA201 cells (33) using a calcium phosphate method (35). When testing for associations between single AChR subunits and chaperones, amounts of cDNAs used were 4 μg of chaperone along with either 1 μgof α or 2 μgof β, γ,or δ.
We examined interactions between the endoplasmic reticulum (ER) chaperones calnexin (CN), ERp57, and immunological heavy chain-binding protein (BiP) and nicotinic acetylcholine receptor (nAChR) subunits. The three chaperones rapidly associate with newly synthesized nAChR subunits. Interactions between nAChR subunits and ERp57 occur via transient intermolecular disulfide bonds and do not require subunit N-linked glycosylation. The associations of ERp57 or CN with AChR subunits are long lived and prolong subunit lifetime ∼10-fold. Coexpression of CN or ERp57 alone does not affect nAChR assembly or trafficking, but together they cause a significant decrease in nAChR expression and assembly. In contrast, associations with BiP are shorter lived and do not alter nAChR expression and assembly. However, a mutated BiP that slows its dissociation significantly increases its associations and decreases nAChR expression and assembly. Our results suggest that interactions with the chaperones regulate the levels of nAChRs assembled in the ER by stabilizing and sequestering subunits during assembly.
N-linked glycosylation is required for nicotinic receptor assembly but not for subunit associations with calnexin
2005, Journal of Biological Chemistry
Citation Excerpt :
All mutants were sequenced to ensure no errors were introduced by the PCR. Transient Transfection—The wild-type and mutant Torpedo AChR subunit cDNAs in the pRBG4 expression vector (30), the canine CN-HA cDNA in the pREP8 vector (Invitrogen, Ref. 31) or the mouse GRP94-FLAG in the pFLAG-CMV-1 vector (Kodak) were transiently transfected into 6-cm cultures of tsA201 cells using a calcium phosphate method (32). Metabolic Labeling, Solubilization, and Subunit Immunoprecipitations—To enhance subunit expression in the mouse L fibroblasts, stably transfected with the Torpedo subunit cDNAs (All-11 cells), cells were grown in DMEM supplemented with 20 mm sodium butyrate 36 h before the experiment.
We investigated how asparagine (N)-linked glycosylation affects assembly of acetylcholine receptors (AChRs) in the endoplasmic reticulum (ER). Block of N-linked glycosylation inhibited AChR assembly whereas block of glucose trimming partially blocked assembly at the late stages. Removal of each of seven glycans had a distinct effect on AChR assembly, ranging from no effect to total loss of assembly. Because the chaperone calnexin (CN) associates with N-linked glycans, we examined CN interactions with AChR subunits. CN rapidly associates with 50% or more of newly synthesized AChR subunits, but not with subunits after maturation. Block of N-linked glycosylation or trimming did not alter CN-AChR subunit associations nor did subunit mutations prevent N-linked glycosylation. Additionally, CN associations with subunits lacking N-linked glycans occurred without subunit aggregation or misfolding. Our data indicate that CN associates with AChR subunits without N-linked glycan interactions. Furthermore, CN-subunit associations only occur early in AChR assembly and have no role in events later that require N-linked glycosylation.
A role for presenilin 1 in regulating the delivery of amyloid precursor protein to the cell surface
2002, Neurobiology of Disease
Presenilin 1 (PS1) and presenilin 2 play a critical role in the γ-secretase processing of amyloid precursor protein (APP) and Notch1. Here, we investigate maturation and intracellular trafficking of APP and other membrane proteins in cells expressing an experimental PS1 deletion mutant (ΔM1,2). Stable expression of ΔM1,2 impairs γ-secretase processing of Notch1 and delays Aβ secretion. Kinetic studies show enhanced O-glycosylation and sialylation of holo-APP and marked accumulation of APP COOH-terminal fragments (CTFs). Surface biotinylation, live staining, and trafficking studies show increased surface accumulation of holo-APP and CTFs in ΔM1,2 cells resulting from enhanced surface delivery of newly synthesized APP. Expression of a loss-of-function PS1 mutant (D385A) or incubation of cells with γ-secretase inhibitors also increases surface levels of holo-APP and CTFs. In contrast to APP, glycosylation and surface accumulation of another type I membrane protein, nicastrin, are markedly reduced in ΔM1,2 cells. Finally, expression of ΔM1,2 results in the increased assembly and surface expression of nicotinic acetylcholine receptors, illustrating that PS1's influence on protein trafficking extends beyond APP and other type I membrane protein substrates of γ-secretase. Collectively, our findings provide evidence that PS1 regulates the glycosylation and intracellular trafficking of APP and select membrane proteins.

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[31] Transient expression of heteromeric ion channels

Publisher Summary

Trends Neurosci.

Methods Enzymol.

J. Biol. Chem.

Cell

Neuron

Methods Enzymol.

Ion Channels: A Practical Approach