Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs.

The glutamate receptor (GluR) channel plays a key part in brain function. Among GluR channel subtypes, the NMDA (N-methyl-D-aspartate) receptor channel which is highly permeable to Ca2+ is essential for the synaptic plasticity underlying memory, learning and development. Furthermore, abnormal activation of the NMDA receptor channel may trigger the neuronal cell death observed in various brain disorders. A complementary DNA encoding a subunit of the rodent NMDA receptor channel (NMDAR1 or zeta 1) has been cloned and its functional properties investigated. Here we report the identification and primary structure of a novel mouse NMDA receptor channel subunit, designated as epsilon 1, after cloning and sequencing the cDNA. The epsilon 1 subunit shows 11-18% amino-acid sequence identity with rodent GluR channel subunits that have been characterized so far and has structural features common to neurotransmitter-gated ion channels. Expression from cloned cDNAs of the epsilon 1 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional GluR channels with high activity and characteristics of the NMDA receptor channel. Furthermore, the heteromeric NMDA receptor channel can be activated by glycine alone.     

Identification by mutagenesis of a Mg(2+)-block site of the NMDA receptor channel.

The N-methyl-D-aspartate (NMDA) receptor channel is highly permeable to Ca2+ but is blocked by Mg2+ in a voltage-dependent manner. These characteristics are essential for the NMDA receptor channel to mediate the induction of long-term potentiation of synaptic efficacy, a form of activity-dependent synaptic plasticity thought to underlie memory, learning and development. Recent studies have revealed the molecular and functional diversity of the NMDA receptor channel subunits, which are classified into the epsilon and zeta families according to the amino-acid sequence homology. Here we report that replacement by glutamine of asparagine 598 in putative transmembrane segment M2 of the zeta 1 subunit, strongly reduces the sensitivity of the heteromeric epsilon 2/zeta 1 NMDA receptor channel to Mg2+ block. The corresponding mutation of the epsilon 2 subunit has a similar effect. Furthermore, the heteromeric epsilon 2/zeta 1 NMDA receptor channel with the mutation on both subunits shows greatly reduced sensitivity to MK-801, a channel blocker of the NMDA receptor channel, but is still susceptible to inhibition by Zn2+. These findings suggest that the conserved asparagine residue in segment M2 constitutes a Mg(2+)-block site of the NMDA receptor channel, and that the MK-801 site overlaps the Mg2+ site.     

Extracellular domains mediating epsilon subunit interactions of muscle acetylcholine receptor.

Ligand-gated ion channels, a major class of cell-surface proteins, have a pseudosymmetric structure with five highly homologous subunits arranged around a central ion pore. The correct assembly of each channel, whose subunit composition varies with cell type and stage of development, requires specific recognition between the subunits. Assembly of the pentameric form of the acetylcholine receptor from adult muscle (AChR; alpha 2 beta epsilon delta) proceeds by a stepwise pathway starting with the formation of the heterodimers, alpha epsilon and alpha delta. The heterodimers than associate with the beta subunit and with each other to form the complete receptor. We have now determined which parts of the subunits mediate the interactions during assembly of the adult form of the receptor from mouse muscle by using a chimaeric subunit in which the N-terminal and C-terminal extracellular domains are derived from the epsilon subunit with the remainder from the beta subunit. The epsilon and beta subunits were chosen because the epsilon subunit forms a heterodimer with the alpha subunit in the pathway for assembly of the receptor, whereas the beta subunit does not. The epsilon beta chimera can substitute for the epsilon but not the beta subunit in the oligomeric receptor, indicating that the alpha subunit specifically recognizes an extracellular domain of the epsilon subunit.     

Subunit arrangement and function in NMDA receptors

Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.     

Acceleration of activation and inactivation by the beta subunit of the skeletal muscle calcium channel.

The L-type voltage-dependent calcium channel is an important link in excitation-contraction coupling of muscle cells (reviewed in refs 2 and 3). The channel has two functional characteristics: calcium permeation and receptor sites for calcium antagonists. In skeletal muscle the channel is a complex of five subunits, alpha 1, alpha 2, beta, gamma and delta. Complementary DNAs to these subunits have been cloned and their amino-acid sequences deduced. The skeletal muscle alpha 1 subunit cDNA expressed in L cells manifests as specific calcium-ion permeation, as well as sensitivity to the three classes of organic calcium-channel blockers. We report here that coexpression of the alpha 1 subunit with other subunits results in significant changes in dihydropyridine binding and gating properties. The available number of drug receptor sites increases 10-fold with an alpha 1 beta combination, whereas the affinity of the dihydropyridine binding site remains unchanged. Also, the presence of the beta subunit accelerates activation and inactivation kinetics of the calcium-channel current.     

NMDA receptors are expressed in developing oligodendrocyte processes and mediate injury

Injury to oligodendrocyte processes, the structures responsible for myelination, is implicated in many forms of brain disorder. Here we show NMDA (N-methyl-D-aspartate) receptor subunit expression on oligodendrocyte processes, and the presence of NMDA receptor subunit messenger RNA in isolated white matter. NR1, NR2A, NR2B, NR2C, NR2D and NR3A subunits showed clustered expression in cell processes, but NR3B was absent. During modelled ischaemia, NMDA receptor activation resulted in rapid Ca2+-dependent detachment and disintegration of oligodendroglial processes in the white matter of mice expressing green fluorescent protein (GFP) specifically in oligodendrocytes (CNP-GFP mice). This effect occurred at mouse ages corresponding to both the initiation and the conclusion of myelination. NR1 subunits were found mainly in oligodendrocyte processes, whereas AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor subunits were mainly found in the somata. Consistent with this observation, injury to the somata was prevented by blocking AMPA/kainate receptors, and preventing injury to oligodendroglial processes required the blocking of NMDA receptors. The presence of NMDA receptors in oligodendrocyte processes explains why previous studies that have focused on the somata have not detected a role for NMDA receptors in oligodendrocyte injury. These NMDA receptors bestow a high sensitivity to acute injury and represent an important new target for drug development in a variety of brain disorders.     

Complete structure and expression in transfected cells of high affinity IgE receptor

The high-affinity receptor for immunoglobulin E, Fc epsilon RI, is found exclusively on mast cells and basophils. When multivalent allergens bind to the receptor-bound IgE, the consequent aggregation of the receptors leads to the release of mediators responsible for allergic symptoms. In rodents Fc epsilon RI is a tetrameric complex of non-covalently attached subunits: one IgE-binding alpha subunit, one beta subunit and a dimer of disulphide-linked gamma subunits. Complementary DNA encoding the alpha and the beta subunits has recently been isolated, but expression of IgE-binding by transfected cells has not yet been achieved. Here we report the cloning of cDNA for the gamma subunit, and propose a model for the alpha beta gamma 2 tetramer which accounts for many of the structural features of the receptor. The rodent receptor on the surface of COS 7 cells was expressed only when the cDNAs for all three subunits were cotransfected. Successful expression of human IgE receptors should now be possible, eventually to permit the detailed analysis of the human IgE-receptor interaction and assist the search for therapeutically effective inhibitors.     

Location of a delta-subunit region determining ion transport through the acetylcholine receptor channel

The combination of complementary DNA expression and single-channel current analysis provides a powerful tool for studying the structure-function relationship of the nicotinic acetylcholine receptor (AChR) (refs 1-5). We have previously shown that AChR channels consisting of subunits from different species, expressed in the surface membrane of Xenopus oocytes, can be used to relate functional properties to individual subunits. Here we report that, in extracellular solution of low divalent cation concentration, the bovine AChR channel has a smaller conductance than the Torpedo AChR channel. Replacement of the delta-subunit of the Torpedo AChR by the bovine delta-subunit makes the channel conductance similar to that of the bovine AChR channel. To locate the region in the delta-subunit responsible for this difference, we have constructed chimaeric delta-subunit cDNAs with different combinations of the Torpedo and bovine counterparts. The conductances of AChR channels containing chimaeric delta-subunits suggest that a region comprising the putative transmembrane segment M2 and the adjacent bend portion between segments M2 and M3 is involved in determining the rate of ion transport through the open channel.     

Induction of calcium currents by the expression of the alpha 1-subunit of the dihydropyridine receptor from skeletal muscle

The dihydropyridine (DHP) receptor purified from skeletal muscle comprises five protein subunits (alpha 1, alpha 2, beta, gamma and delta) and produces Ca2+ currents that are blocked by DHPs. Cloning of the alpha 1- and alpha 2-subunits, the former affinity-labelled by DHP, has shown that the alpha 1-subunit is expressed in skeletal muscle alone, whereas the alpha 2- and delta- subunits are also expressed in other tissues. Although the transient expression of the alpha 1-subunit in myoblasts from dysgenic mice (but not in oocytes) has been demonstrated, the use of these expression systems to determine the function of the alpha 1- subunit is complicated by the presence of endogenous Ca2+ currents, which may reflect the constitutive expression of proteins similar to the alpha 2-, beta-, gamma- and/or delta-subunits. We therefore selected a cell line which has no Ca2+ currents or alpha 2- subunit, and probably no delta-subunit for stable transformation with complementary DNA of the alpha 1- subunit. The transformed cells express DHP-sensitive, voltage-gated Ca2+ channels, indicating that the minimum structure of these channels is at most an alpha 1 beta gamma complex and possibly an alpha 1- subunit alone.     

Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor

Neuronal nicotinic acetylcholine receptors are members of a gene family of ligand-gated transmitter receptors that includes muscle nicotinic receptors, GABAA receptors and glycine receptors. Several lines of evidence indicate that neuronal nicotinic receptors can be made up of only two subunits, an alpha (alpha) subunit which binds ligand, and a non-alpha (n alpha) or beta (beta) subunit. The stoichiometry of each subunit in the functional receptor has been difficult to assess, however. Estimates of the molecular weight of neuronal nicotonic receptor macromolecules suggest that these receptors contain at least four subunits but probably not more than five. We have examined the subunit stoichiometry of the chick neuronal alpha 4/n alpha 1 receptor by first using site-directed mutagenesis to create subunits that confer different single channel properties on the receptor. Co-injection with wild-type and mutant subunits led to the appearance of receptors with wild-type, mutant and hybrid conductances. From the number of hybrid conductances, we could deduce the number of each subunit in the functional receptor.     

Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA.

Fast excitatory transmission in the vertebrate central nervous system is mediated mainly by L-glutamate. On the basis of pharmacological, physiological and agonist binding properties, the ionotropic glutamate receptors are classified into NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate) and kainate subtypes. Sequence homology between complementary DNA clones encoding non-NMDA glutamate receptor subunits reveals at least two subunit classes: the GluR1 to GluR4 class and the GluR5 class. Here we report the cloning and expression of a functional rat glutamate receptor subunit cDNA, GluR6, which has a very different pharmacology from that of the GluR1-GluR4 class. Receptors generated from the GluR1-GluR4 class have a higher apparent affinity for AMPA than for kainate. When expressed in Xenopus oocytes the homomeric GluR6 receptor is activated by kainate, quisqualate and L-glutamate but not by AMPA, and the apparent affinity for kainate is higher than for receptors from the GluR1-GluR4 class. Desensitization of the receptor was observed with continuous application of agonist. The homomeric GluR6 glutamate receptor exhibits an outwardly rectifying current-voltage relationship. In situ hybridizations reveal a pattern of GluR6 gene expression reminiscent of the binding pattern obtained with [3H]kainate.     

Cloning of cDNA for the glutamate-binding subunit of an NMDA receptor complex.

The amino acids L-glutamic and L-aspartic acids form the most widespread excitatory transmitter network in mammalian brain. The excitation produced by L-glutamic acid is important in the early development of the nervous system, synaptic plasticity and memory formation, seizures and neuronal degeneration. The receptors activated by L-glutamic acid are a target for therapeutic intervention in neurodegenerative diseases, brain ischaemia and epilepsy. There are two types of receptors for the excitatory amino acids, those that lead to the opening of cation-selective channels and those that activate phospholipase C (ref. 11). The receptors activating ion channels are NMDA (N-methyl-D-aspartate) and kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive receptors. The complementary DNAs for the kainate/AMPA receptor and for the metabotropic receptor have been cloned. We report here on the isolation and characterization of a protein complex of four major proteins that represents an intact complex of the NMDA receptor ion channel and on the cloning of the cDNA for one of the subunits of this receptor complex, the glutamate-binding protein.     

Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits

The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves critical functions in physiological and pathological processes in the central nervous system, including neuronal development, plasticity and neurodegeneration. Conventional heteromeric NMDARs composed of NR1 and NR2A-D subunits require dual agonists, glutamate and glycine, for activation. They are also highly permeable to Ca2+, and exhibit voltage-dependent inhibition by Mg2+. Coexpression of NR3A with NR1 and NR2 subunits modulates NMDAR activity. Here we report the cloning and characterization of the final member of the NMDAR family, NR3B, which shares high sequence homology with NR3A. From in situ and immunocytochemical analyses, NR3B is expressed predominantly in motor neurons, whereas NR3A is more widely distributed. Remarkably, when co-expressed in Xenopus oocytes, NR3A or NR3B co-assembles with NR1 to form excitatory glycine receptors that are unaffected by glutamate or NMDA, and inhibited by D-serine, a co-activator of conventional NMDARs. Moreover, NR1/NR3A or -3B receptors form relatively Ca2+-impermeable cation channels that are resistant to Mg2+, MK-801, memantine and competitive antagonists. In cerebrocortical neurons containing NR3 family members, glycine triggers a burst of firing, and membrane patches manifest glycine-responsive single channels that are suppressible by D-serine. By itself, glycine is normally thought of as an inhibitory neurotransmitter. In contrast, these NR1/NR3A or -3B 'NMDARs' constitute a type of excitatory glycine receptor.     

NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones

Excitatory amino acids act via receptor subtypes in the mammalian central nervous system (CNS). The receptor selectively activated by N-methyl-D-aspartic acid (NMDA) has been best characterized using voltage-clamp and single-channel recording; the results suggest that NMDA receptors gate channels that are permeable to Na+, K+ and other monovalent cations. Various experiments suggest that Ca2+ flux is also associated with the activation of excitatory amino-acid receptors on vertebrate neurones. Whether Ca2+ enters through voltage-dependent Ca2+ channels or through excitatory amino-acid-activated channels of one or more subtype is unclear. Mg2+ can be used to distinguish NMDA-receptor-activated channels from voltage-dependent Ca2+ channels, because at micromolar concentrations Mg2+ has little effect on voltage-dependent Ca2+ channels while it enters and blocks NMDA receptor channels. Marked differences in the potency of other divalent cations acting as Ca2+ channel blockers compared with their action as NMDA antagonists also distinguish the NMDA channel from voltage-sensitive Ca2+ channels. However, we now directly demonstrate that excitatory amino acids acting at NMDA receptors on spinal cord neurones increase the intracellular Ca2+ activity, measured using the indicator dye arsenazo III, and that this is the result of Ca2+ influx through NMDA receptor channels. Kainic acid (KA), which acts at another subtype of excitatory amino-acid receptor, was much less effective in triggering increases in intracellular free Ca2+.     

Cerebellar GABAA receptor selective for a behavioural alcohol antagonist

Benzodiazepines are widely prescribed anxiolytics and anticonvulsants which bind with high affinity to sites on the GABAA receptor/Cl- channel complex and potentiate the effect of the neurotransmitter GABA (gamma-aminobutyric acid). The heterogeneity of benzodiazepine recognition sites in the central nervous system was revealed by studies showing different classes of GABAA receptor subunits (classes alpha, beta and gamma) and variant subunits in these classes, particularly in the alpha-class. Expression of recombinant subunits produces functional receptors; when certain alpha-variants are coexpressed with beta- and gamma-subunits the resulting receptors have pharmacological properties characteristic of GABAA-benzodiazepine type I or type II receptors. The alpha-variants are differentially expressed in the central nervous system and can be photoaffinity-labelled with benzodiazepines. Here we report a novel alpha-subunit (alpha 6) of cerebellar granule cells. We show that recombinant receptors composed of alpha 6, beta 2 and gamma 2 subunits bind with high affinity to the GABA agonist [3H]muscimol and the benzodiazepine [3H]Ro15-4513 but not the other benzodiazepines or beta-carboniles. The same distinctive pharmacology is observed with GABAA receptors from rat cerebellum immunoprecipitated by an antiserum specific for the alpha 6 subunit. We conclude that this alpha-subunit is part of a cerebellar receptor subtype, selective for Ro15-4513, an antagonist of alcohol-induced motor incoordination and ataxia.     

Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases.

The high-affinity IgE receptor (Fc epsilon RI), which is expressed on the surface of mast cells and basophils, has a central role in immediate allergic responses. In the rat basophilic leukaemia cell line RBL-2H3, which is a model system for the analysis of Fc epsilon RI-mediated signal transduction, surface engagement of Fc epsilon RI induces histamine release and the tyrosine phosphorylation of several distinct proteins. Although the alpha, beta, and gamma subunits of Fc epsilon RI lack intrinsic tyrosine protein kinase (TPK) activity, a kinase that copurifies with Fc epsilon RI phosphorylates the beta and gamma subunits of the receptor on tyrosine residues. We report here that in RBL-2H3 cells, p56lyn and pp60c-src are activated after Fc epsilon RI crosslinking, and p56lyn coimmunoprecipitates with Fc epsilon RI. In the mouse mast-cell line PT-18, another cell type used to study FC epsilon RI-mediated signalling, tyrosine phosphorylation of proteins is also an immediate consequence of receptor crosslinking. Notably, the only detectable src protein-related TPK in PT-18 cells is p62c-yes, and it is this TPK that is activated on Fc epsilon RI engagement and coimmunoprecipitates with the receptor. Therefore, it seems that different src protein-related TPKs can associate with the same receptor and become activated after receptor engagement.     

A cytoplasmic region determines single-channel conductance in 5-HT3 receptors

5-hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the 'HA-stretch') within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.     

Potentiation of NMDA receptor currents by arachidonic acid.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.     

Vasoregulation by the beta1 subunit of the calcium-activated potassium channel

Small arteries exhibit tone, a partially contracted state that is an important determinant of blood pressure. In arterial smooth muscle cells, intracellular calcium paradoxically controls both contraction and relaxation. The mechanisms by which calcium can differentially regulate diverse physiological responses within a single cell remain unresolved. Calcium-dependent relaxation is mediated by local calcium release from the sarcoplasmic reticulum. These 'calcium sparks' activate calcium-dependent potassium (BK) channels comprised of alpha and beta1 subunits. Here we show that targeted deletion of the gene for the beta1 subunit leads to a decrease in the calcium sensitivity of BK channels, a reduction in functional coupling of calcium sparks to BK channel activation, and increases in arterial tone and blood pressure. The beta1 subunit of the BK channel, by tuning the channel's calcium sensitivity, is a key molecular component in translating calcium signals to the central physiological function of vasoregulation.     

The heteromeric cyclic nucleotide-gated channel adopts a 3A:1B stoichiometry

Cyclic nucleotide-gated (CNG) channels are crucial for visual and olfactory transductions. These channels are tetramers and in their native forms are composed of A and B subunits, with a stoichiometry thought to be 2A:2B (refs 6, 7). Here we report the identification of a leucine-zipper-homology domain named CLZ (for carboxy-terminal leucine zipper). This domain is present in the distal C terminus of CNG channel A subunits but is absent from B subunits, and mediates an inter-subunit interaction. With cross-linking, non-denaturing gel electrophoresis and analytical centrifugation, this CLZ domain was found to mediate a trimeric interaction. In addition, a mutant cone CNG channel A subunit with its CLZ domain replaced by a generic trimeric leucine zipper produced channels that behaved much like the wild type, but less so if replaced by a dimeric or tetrameric leucine zipper. This A-subunit-only, trimeric interaction suggests that heteromeric CNG channels actually adopt a 3A:1B stoichiometry. Biochemical analysis of the purified bovine rod CNG channel confirmed this conclusion. This revised stoichiometry provides a new foundation for understanding the structure and function of the CNG channel family.