Xenopus Oocytes express a unitary glutamate receptor endogenously

doi:10.1006/jmbi.1997.1272

Journal of Molecular Biology

Volume 273, Issue 1, 17 October 1997, Pages 14-18

https://doi.org/10.1006/jmbi.1997.1272 Get rights and content

Abstract

The results described here demonstrate that Xenopus oocytes endogenously express a unitary glutamate receptor subunit XenU1. The level of XenU1 mRNA expression reaches approximately 1/300 of that in the adult Xenopus brain. The endogenous expression of XenU1, which can functionally interact with N-methyl-d-aspartate receptor subunit NR1, explains the differences in NR1 subunit expression in mammalian cell lines (no functional expression without partner subunits) and in the Xenopus oocytes (NR1 forms functional receptors when expressed singly).

References (29)

M. Hollmann et al.
Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor
Neuron
(1993)
K. Ikeda et al.
Cloning and expression of the epsilon4 subunit of the NMDA receptor channel
FEBS Letters
(1992)
T. Ishii et al.
Molecular characterization of the family of the N-methyl-d-aspartate receptor subunits
J. Biol. Chem.
(1993)
A. Kuryatov et al.
Mutational analysis of the glycine-binding site of the NMDA receptorstructural similarity with bacterial amino acid-binding proteins
Neuron
(1994)
B. Laube et al.
Molecular determinants of agonist discrimination by NMDA receptor subunitsanalysis of the glutamate binding site on the NR2B subunit
Neuron
(1997)
D.J. Laurie et al.
Ligand affinities at recombinant N-methyl-d-aspartate receptors depend on subunit composition
Eur. J. Pharmacol.
(1994)
M.M. Soloviev et al.
Functional expression of a recombinant unitary glutamate receptor from Xenopus, which contains N-methyl-d-aspartate (NMDA) and non-NMDA receptor subunits
J. Biol. Chem.
(1996)
N.J. Sucher et al.
NMDA receptorsfrom genes to channels
Trends Pharmacol. Sci.
(1996)
P.L. Chazot et al.
Immunological detection of the NMDAR1 glutamate receptor subunit expressed in human embryonic kidney 293 cells and in rat brain
J. Neurochem.
(1992)
M. Cik et al.
Expression of NMDAR1-1a (N598Q)/NMDAR2A receptors results in decreased cell mortality
Eur. J. Pharmacol.
(1994)

G.M. Durand et al.

Splice variants of the N-methyl-d-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C

Proc. Natl Acad. Sci. USA

(1993)

S. Grimwood et al.

Recombinant human NMDA homomeric NR1 receptors expressed in mammalian cells form a high-affinity glycine antagonist binding site

J. Neurochem.

(1995)

J.B. Gurdon et al.

Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells

Nature

(1971)

J.M. Henley

Characterization of the allosteric modulatory protein associated with non-NMDA receptors

Biochem. Soc. Trans.

(1993)

Cited by (39)

Cave Canalem: How endogenous ion channels may interfere with heterologous expression in Xenopus oocytes
2010, Methods
Citation Excerpt :
In contrast to other heterologous expression systems, in Xenopus oocytes the formation of functional homomeric NR1 receptors had been reported for decades [86], in spite of the fact that the putatively homomeric receptors displayed properties which could not be explained by one type of subunit alone [87]. The postulate that exogenous NR1 subunits assemble with the endogenous kainate binding protein XenU1 [88] had been the accepted explanation for the formation of functional NMDA receptors upon homomeric expression of exogenous NR1 in oocytes for a long time. This explanation had to be revised in the light of electrophysiological and pharmacological evidence that the postulated NR1/XenU1 receptors could not be identical to the receptors formed upon expression of NR1 alone [89,90].
Xenopus laevis oocytes are an outstanding heterologous expression system for the investigation of ion channels. However, oocytes express an amazing variety of endogenous ion channels that can severely interfere with electrophysiological measurements. It is therefore necessary to be aware of the channels present in the oocyte and to be able to exclude artifacts they might cause during the analysis of heterologously expressed ion channels. Research on Xenopus endogenous ion channels has started over 30 years ago, and many channels have been described since then. This does not only include voltage-gated channels conducting Na⁺, K⁺, Ca²⁺, and Cl⁻, but also ion channels activated by ligand binding such as ionotropic neurotransmitter receptors. Furthermore, there are other channels such as those triggered by changes in osmolarity or mechanical stress, as well as conductances caused by yet uncharacterized molecules. Here, we present an overview of ion channels endogenous to the oocyte described in the literature so far, and provide procedures and methods to abolish or minimize their impact on electrophysiological recordings of exogenous channels.
Xenopus laevis Oocytes Endogenously Express All Subunits of the Ionotropic Glutamate Receptor Family
2009, Journal of Molecular Biology
Xenopus laevis oocytes are commonly used as a heterologous expression system for the electrophysiological characterization of ionotropic glutamate receptors (iGluRs). Recently, however, several glutamate receptor subunits of the N-methyl-d-aspartate receptor subfamily have been found to be expressed endogenously in Xenopus oocytes, thus limiting the use of this expression system for such receptors. We therefore screened oocytes for the Xenopus homologs of all iGluR subunits known to be expressed in mammals to investigate which additional subunits may be present endogenously in oocytes and what, if any, influence such proteins might have on the functional analysis of heterologously expressed receptors. We found Xenopus homologs of every mammalian iGluR subunit to be expressed at the mRNA level. We then cloned, from oocytes, full-length copies of the four Xenopus α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunits XenGluR1 through XenGluR4 and, additionally, XenGluR6 as a representative subunit for the kainate receptor subfamily and electrophysiologically characterized them. Upon analysis, we found only minor functional differences between homologous subunits from Xenopus and rat. Next, we investigated whether endogenous iGluR subunits can be detected electrophysiologically in oocytes. We found no indication for any functional glutamate receptors in native oocytes; however, after heterologous expression of the auxiliary subunit stargazin, endogenous α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors were detected. These data demonstrate that Xenopus oocytes express glutamate receptor subunits endogenously, albeit at very low levels. Such endogenous receptor proteins can, under certain circumstances, become electrophysiologically detectable and then might influence electrophysiological recordings performed on recombinant receptors in oocytes.
Apparent Homomeric NR1 Currents Observed in Xenopus Oocytes are Caused by an Endogenous NR2 Subunit
2008, Journal of Molecular Biology
Functional N-methyl-d-aspartate receptors NMDARs are thought to be heteromeric receptor complexes consisting of NR1 and NR2 subunits. However, recombinant NR1 subunits expressed in Xenopus oocytes assemble functional ion channels even without exogenous NR2 subunits and with a different pharmacology, suggesting a homomeric subunit stoichiometry. To explain this phenomenon, we screened oocytes for Xenopus NR2 subunits and found all four subunit-encoding mRNAs (XenNR2A–XenNR2D) to be present endogenously, with those encoding the XenNR2B subunit being particularly abundant. We cloned the full-length XenNR2B cDNA and co-expressed it with NR1 in oocytes. A detailed electrophysiological characterization revealed that the pharmacology of NR1/XenNR2B was identical with that of the presumed homomeric NMDARs expressed from NR1 subunits. By contrast, heteromeric receptors containing the rat NR2B subunit showed significant pharmacological differences compared with NR1/XenNR2B receptors. These results demonstrate that recombinant NR1 subunits expressed in Xenopus oocytes interact with an endogenously expressed NR2B subunit and form hybrid heteromeric NMDARs. These findings confirm the current views that NMDARs are obligatory heteromeric complexes and that functional homomeric NMDARs do not exist.
Kainate-binding proteins are rendered functional ion channels upon transplantation of two short pore-flanking domains from a kainate receptor
2002, Journal of Biological Chemistry
Citation Excerpt :
Obvious explanations such as different concentrations of injected RNA or different protein expression levels can be excluded because these parameters were controlled for and monitored, respectively. We can also exclude a varying contribution by the endogenous Xenopus kainate-binding protein XenU1 (7, 24) being responsible for this effect. Coexpression of the functional chimeras KBP-(linkerA+B)GluR6 and KBP-PCL-PXL with recombinantXenopus KBP (7) did not increase the probability of current expression.
Kainate-binding proteins belong to an elusive class of putative ionotropic glutamate receptors that to date have not been shown to form functional ion channels in heterologous expression systems, despite binding glutamatergic agonists with high affinity. To test the hypothesis that inefficient or interrupted signal transduction from the ligand-binding site via linker domains to the ion pore (gating) might be responsible for this apparent lack of function, we transplanted the short homologous linker sequences from the fully functional rat kainate receptor GluR6 into frog kainate-binding protein. We were able to generate chimeric receptors that are functional in the Xenopus oocyte expression system and in human embryonic kidney 293 cells. The linker domains A and B in particular appear to be crucial for gating, because a functional kainate-binding protein was observed when at least parts of both linkers were derived from GluR6. We speculate that to enable signal transduction from the ligand-binding site to the ion pore of the frog kainate-binding protein, the linker structure of the protein has to undergo an essential conformational alteration, possibly mediated by an as yet unknown subunit or modulatory protein.
Ethanol sensitivity of NMDA receptors
2002, Neurochemistry International
NMDA receptors are ionotropic glutamate receptors assembled of subunits of the NR1 and of the NR2 family (NR2A–NR2D). The subunit diversity largely affects the pharmacological properties of NMDA receptors and, hence, gives rise to receptor heterogeneity. As an overall result of studies on recombinant and native NMDA receptors, ethanol inhibits the function of receptors containing the subunits NR2A and/or NR2B to a greater extent than those containing NR2C or NR2D. For example, in rat cultured mesencephalic neurons, NR2C expression was developmentally increased, whereas expression of NR2A and NR2B was decreased. These changes coincided with a developmental loss of sensitivity of NMDA responses to ethanol and ifenprodil, a non-competitive NMDA receptor antagonist that shows selectivity for NR2B-containing receptors. Also in rat locus coeruleus neurons, the low ethanol sensitivity of somatic NMDA receptors could be explained by a prominent expression of NR2C. The inhibitory site of action for ethanol on the NMDA receptor is not yet known. Patch–clamp studies suggest a target site exposed to or only accessible from the extracellular environment. Apparently, amino acid residue Phe⁶³⁹, located in the TM3 domain of NR1, plays a crucial role in the inhibition of NMDA receptor function by ethanol. Since this phenylalanine site is common to all NMDA and non-NMDA receptor (AMPA/kainate receptor) subunits, this observation is consistent with accumulating evidence for a similar ethanol sensitivity of a variety of NMDA and non-NMDA receptors, but it cannot explain the differences in ethanol sensitivity observed with different NR2 subunits.
Identification of a new site in the S1 ligand binding region of the NMDA receptor NR2A subunit involved in receptor activation by glutamate
2002, Neuropharmacology
Activation of N-methyl-d-aspartate (NMDA) receptors requires the binding of both glutamate and glycine to independent sites on the receptor. These ligands bind to NR2 and NR1 subunits respectively. Ligand binding residues are located in two non-contiguous domains, S1 and S2, which have been implicated in glutamate binding in other ionotropic glutamate receptor subunits. To further define the amino acids through which glutamate activates the receptor, we generated single-site mutations to the NR2A subunit, and expressed them with wild type NR1 in HEK 293 cells. Using calcium imaging and whole cell patch clamp we determined glutamate and glycine potencies. Of the eight residues mutated we identified five (E413, K484, A508, G685 and G688), whose mutation leads to a large reduction (from 4- to 1000-fold) in glutamate potency, consistent with a role for these residues in receptor activation by glutamate. The potency of glycine was largely unchanged by these mutations. Thus our results extend the knowledge base of residues involved in NMDA receptor function and identifies a new site in S1, in the region of A508, that has a role in receptor activation by glutamate.

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¹: Edited by W. Baumeister

²: Correspondence address: M. M. Soloviev, c/o E. A. Barnard, Molecular Neurobiology Unit, Royal Free Hospital School of Medicine, London NW3 2PF, UK.

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Abstract

References (29)

Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor

Neuron

Cloning and expression of the epsilon4 subunit of the NMDA receptor channel

FEBS Letters

Molecular characterization of the family of the N-methyl-d-aspartate receptor subunits

J. Biol. Chem.

Mutational analysis of the glycine-binding site of the NMDA receptorstructural similarity with bacterial amino acid-binding proteins

Neuron

Molecular determinants of agonist discrimination by NMDA receptor subunitsanalysis of the glutamate binding site on the NR2B subunit

Neuron

Ligand affinities at recombinant N-methyl-d-aspartate receptors depend on subunit composition

Eur. J. Pharmacol.

Functional expression of a recombinant unitary glutamate receptor from Xenopus, which contains N-methyl-d-aspartate (NMDA) and non-NMDA receptor subunits

J. Biol. Chem.

NMDA receptorsfrom genes to channels

Trends Pharmacol. Sci.

Immunological detection of the NMDAR1 glutamate receptor subunit expressed in human embryonic kidney 293 cells and in rat brain

J. Neurochem.

Expression of NMDAR1-1a (N598Q)/NMDAR2A receptors results in decreased cell mortality

Eur. J. Pharmacol.

Splice variants of the N-methyl-d-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C

Proc. Natl Acad. Sci. USA

Recombinant human NMDA homomeric NR1 receptors expressed in mammalian cells form a high-affinity glycine antagonist binding site

J. Neurochem.

Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells

Nature

Characterization of the allosteric modulatory protein associated with non-NMDA receptors

Biochem. Soc. Trans.

Cited by (39)

Cave Canalem: How endogenous ion channels may interfere with heterologous expression in Xenopus oocytes

Xenopus laevis Oocytes Endogenously Express All Subunits of the Ionotropic Glutamate Receptor Family

Apparent Homomeric NR1 Currents Observed in Xenopus Oocytes are Caused by an Endogenous NR2 Subunit

Kainate-binding proteins are rendered functional ion channels upon transplantation of two short pore-flanking domains from a kainate receptor

Ethanol sensitivity of NMDA receptors

Identification of a new site in the S1 ligand binding region of the NMDA receptor NR2A subunit involved in receptor activation by glutamate

Journal of Molecular Biology

CommunicationXenopus Oocytes express a unitary glutamate receptor endogenously1

Abstract

Neuron

FEBS Letters

J. Biol. Chem.

Neuron

Neuron

Eur. J. Pharmacol.

J. Biol. Chem.

Trends Pharmacol. Sci.

Immunological detection of the NMDAR1 glutamate receptor subunit expressed in human embryonic kidney 293 cells and in rat brain

J. Neurochem.

Expression of NMDAR1-1a (N598Q)/NMDAR2A receptors results in decreased cell mortality

Eur. J. Pharmacol.

Splice variants of the N-methyl-d-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C

Proc. Natl Acad. Sci. USA

Recombinant human NMDA homomeric NR1 receptors expressed in mammalian cells form a high-affinity glycine antagonist binding site

J. Neurochem.

Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells

Nature

Characterization of the allosteric modulatory protein associated with non-NMDA receptors

Biochem. Soc. Trans.

Communication
Xenopus Oocytes express a unitary glutamate receptor endogenously¹