Biomedical subjects

G L Westbrook

Publications and source records attributed to G L Westbrook.

At least 19 recordsLinked to original sources

PubMed1998

Activation kinetics of AMPA receptor channels reveal the number of functional agonist binding sites.

AMPA and NMDA receptor channels are closely related molecules, yet they respond to glutamate with distinct kinetics, attributable to differences in ligand binding and channel gating steps (for review, see Edmonds et al., 1995). We used two complementary approaches to investigate the number of functional binding sites on AMPA channels on outside-out patches from cultured hippocampal neurons. The activation kinetics of agonist binding were measured during rapid steps into low concentrations of selective AMPA receptor agonists and during steps from a competitive AMPA receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione, into a saturating concentration of agonist. Both approaches revealed sigmoidal kinetics, which suggests that multiple agonist binding steps or antagonist unbinding steps are needed for channel activation. A kinetic model with two independent binding sites gave a better fit to the activation phase than models with one or three independent sites. A more refined analysis incorporating cooperative interaction between the two binding sites significantly improved the fits to the responses. The affinity of the first binding step was two to three times higher than the second step. These results demonstrate that binding of two agonist molecules are needed to activate AMPA receptors, but the two binding sites are not identical and independent. Because NMDA receptors require four ligand molecules for activation (two glycine and two glutamate; Benveniste and Mayer, 1991; Clements and Westbrook, 1991), it may be that some binding sites on AMPA receptors are functionally silent.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1997

Shaping of IPSCs by endogenous calcineurin activity.

Synaptic inhibition, mediated by GABAA receptors, regulates neuronal firing, influences coincidence detection (König et al., 1996), and can synchronize the output of neural circuits (Cobb et al., 1995). Although GABAA receptors can be modulated by phosphorylation, few studies have directly addressed the role of such modulation at synapses, where the nonequilibrium conditions of receptor activation are quite different from those often used to study GABAA receptors in vitro. Here we promoted endogenous phosphorylation by inhibiting specific phosphatases in rat hippocampal neurons and compared the effects on IPSCs with GABAA channel responses in outside-out patches. Brief and saturating GABA pulses (5 msec; 10 mM) activated patch currents resembling the IPSC. Inhibition of calcineurin (protein phosphatase 2B), but not phosphatases 1 or 2A, produced a similar shortening of IPSC and patch responses, as did nonspecific inhibition of dephosphorylation using ATPgammaS or high concentrations of intracellular phosphate. Calcineurin inhibition increased the microscopic ligand unbinding rate, which was measured using the competitive antagonist 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide, suggesting that the IPSC shortening was partly caused by destabilization of the ligand binding site. Calcineurin inhibition also increased the rate and extent of macroscopic receptor desensitization. These results show that endogenous regulation by kinases and calcineurin can produce substantial changes in the IPSC duration by altering the unbinding and gating kinetics of the GABAA receptor. Dynamic regulation of synaptic inhibition may thus allow for the tuning of circuit behavior at the level of individual inhibitory synapses.

Animals↗

PubMed1997

Immunolocalization of metabotropic glutamate receptor 7 in the rat olfactory bulb.

Metabotropic glutamate receptors (mGluRs) constitute a large family of G-protein-coupled receptors that are subdivided into three groups based on sequence similarity, pharmacological profiles, and coupling to second messengers. Although mRNAs for seven of the eight mGluRs are expressed in the olfactory system, the localization and function of specific subtypes have not been fully characterized. Mitral cells of the olfactory bulb express mRNA for several mGluRs, including mGluR7, which has been suggested as a presynaptic glutamate autoreceptor. To investigate the immunolocalization of mGluR7 in the olfactory system, we used a polyclonal antiserum specific for the carboxy terminus of the receptor. Mitral cell somata and proximal dendrites were strongly labeled by the mGluR7 antibody. Electron microscopic analysis revealed that most of the mitral cell somatic staining was cytoplasmic. In olfactory bulb glomeruli, immunoreactivity was present in axons and dendrites. In the piriform cortex, diffuse staining was present in layer Ia that was markedly reduced following bulbectomy, consistent with expression of mGluR7 in mitral cell axon terminals. Electron microscopic analysis of this region confirmed the presence of mGluR7 in multiple axon terminals. Distinct labeled fibers in all levels of layer I appeared to originate from labeled piriform cortex pyramidal cells in layers II and III. Our results indicate that mGluR7 is primarily presynaptic at olfactory bulb synapses. However, the postsynaptic localization of mGluR7 at selected synapses indicates that mGluR7 is not targeted exclusively to axonal compartments.

Animals↗

PubMed1997

Cloning and expression of rat metabotropic glutamate receptor 8 reveals a distinct pharmacological profile.

The metabotropic glutamate receptor (mGluR) cDNAs were originally cloned from rat, except for the mouse cDNA clone encoding mGluR8. Mouse mGluR8 couples weakly to the inhibition of adenylate cyclase, thus hindering the characterization of its pharmacological properties. We isolated a rat mGluR8 cDNA that encodes a protein of 908 amino acids. In situ hybridization revealed prominent mGluR8 mRNA expression in olfactory bulb, pontine gray, lateral reticular nucleus of the thalamus, and piriform cortex. Less abundant expression was detected in cerebral cortex, hippocampus, cerebellum, and mammillary body. Glutamate evoked pertussis toxin-sensitive potassium currents in Xenopus laevis oocytes coexpressing mGluR8 and G protein-coupled inwardly rectifying potassium channels. mGluR8 was also activated by the group III-specific agonist L-2-amino-4-phosphonobutyric acid; (2(S), 1'(S), 2'(S)]- 2-(carboxycyclopropyl)glycine, which has been frequently used as a selective group II agonist; and the nonselective agonist (1(S), 3(R)]-1-aminocyclopentane-1,3-dicarboxylic acid but not by the group I-specific agonist 3,5-dihydroxyphenylglycine or the group II-specific agonist [2(S), 1'(R), 2(R), 3'(R)]-2-(2, 3-dicarboxycyclopropyl)glycine. The agonist profile in order of potency was [2(S), 1'(S), 2'(S)]-2-(carboxycyclopropyl)glycine approximately L-2-amino-4-phosphonobutyric acid > glutamate > > [1(S), 3(R)]-1-aminocyclopentane-1, 3-dicarboxylic acid, with EC50 values of 0.63, 0.67, 2.5, and 47 microM, respectively. Both the group I/II-specific antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine and the group III-specific antagonist alpha-methyl-amino-phosphonobutyrate inhibited mGluR8. The pharmacological profile of mGluR8 is distinct among mGluRs but closely matches that of presynaptic inhibition in some central nervous system pathways. Thus, cellular responses mediated by both group II and III agonists may in some cases reflect activation of mGluR8 rather than multiple mGluR subtypes.

Amino Acid Sequence↗

PubMed1997

Evaluation of Vitek GNI+ and Becton Dickinson Microbiology Systems Crystal E/NF identification systems for identification of members of the family Enterobacteriaceae and other gram-negative, glucose-fermenting and non-glucose-fermenting bacilli.

We evaluated the Vitek GNI+ and Becton Dickinson Crystal E/NF identification systems for their ability to accurately identify 619 and 626 strains, respectively, of members of the family Enterobacteriaceae and other glucose-fermenting and non-glucose-fermenting gram-negative rods. All strains tested were taken from a stock collection and passed three times on 5% sheep blood agar prior to testing. These strains represented a more rigorous challenge to both systems than one resulting from the testing of consecutive clinical isolates. Testing with both systems was done according to the manufacturers' instructions, and tests were repeated in duplicate when errors occurred. Vitek version 5.01 and Crystal version 3.0 softwares were used for identifications. The identification results from each system were compared with identifications previously determined with reference biochemicals. At the completion of the appropriate incubation period, the GNI+ and Crystal systems correctly identified 80.1 and 71.1% of the total isolates, respectively. After additional tests suggested by the software programs were completed, the GNI+ had an accuracy of 87.6% and the Crystal system's accuracy had improved to 87.9%. The error rates for the GNI+ and Crystal systems were 6.5 and 5.3%, respectively. A report of "no identification" was given for 6.0 and 6.9% of the isolates, respectively, and was associated with no particular organism group. One isolate each of Acinetobacter lwoffii and Vibrio alginolyticus would not grow in the Vitek card. The average times to detection for correct enteric identifications in the GNI+ system were 4.1 and 6.8 h for nonenteric identifications, while the Crystal results were routinely read at 18 h. We conclude that there was no significant difference (P > 0.05) between the results of the GNI+ card and those of the Crystal E/NF system after additional testing was performed with the group of organisms tested, but the overall accuracy for both systems in this study was below 90%.

Bacterial Typing Techniques↗

PubMed1997

Modulation of mEPSCs in olfactory bulb mitral cells by metabotropic glutamate receptors.

Olfactory bulb mitral cells express group I (mGluR1), group II (mGluR2), and group III (mGluR7 and mGluR8) metabotropic glutamate receptors. We examined the role of these mGluRs on excitatory synaptic transmission in cultured mitral cells with the use of whole cell patch-clamp recordings. The effects of group-selective mGluR agonists and antagonists were tested on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor-mediated miniature excitatory postsynaptic currents (mEPSCs). (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylate (ACPD) or the group-I-selective agonist 3,5-dihydroxyphenylglycine evoked an inward current accompanied by a decrease in membrane conductance, consistent with the previously described closure of potassium channels by group I agonists. The increased cellular excitability was accompanied by an increase in mEPSC frequency in some cells. When calcium entry was blocked by cadmium, ACPD or the group-II-selective agonist 2-(2,3-dicarboxycyclopropyl)-glycine reduced the mEPSC frequency. L-2-amino-4-phosphonobutyric acid (L-AP4), a group-III-selective agonist, caused a similar decrease. The concentration-dependence of L-AP4-mediated inhibition was most consistent with activation of mGluR8. We investigated two possible effector mechanisms for the group III presynaptic receptor. Bath application of forskolin or 3-isobutyl-1-methylxantine had no effect on mEPSC frequency. Increasing calcium influx by raising extracellular K+ caused a large increase in the mEPSC frequency but did not enhance L-AP4-mediated inhibition. Thus inhibition of mEPSCs involves a mechanism downstream of calcium entry and appears to be independent of adenosine 3',5'-cyclic monophosphate. Our results indicate that both group II and III receptors can inhibit glutamate release at mitral cell terminals. Although group II/III receptors had a similar effect on mEPSCs, differences in location on nerve terminals and in glutamate sensitivity suggest that each mGluR may have discrete actions on mitral cell activity.

Adenylyl Cyclases↗

PubMed1997

Cytoskeletal interactions with glutamate receptors at central synapses.

The data presented here are clearly just the beginning of any comprehensive understanding of the set of regulatory and cytoskeletal proteins that interact with membrane receptors in the postsynaptic density. They do, however, indicate that both glutamate channels at central excitatory synapses are involved in complex protein-protein interactions. For example, while NR2A is important for Ca-dependent inactivation of NMDA receptors, studies in several systems suggest that the other major NR2 subunit in hippocampal neurons, NR2B, predominates at critical times during synapse formation. In addition, the COOH terminus of NR2B binds to several novel cytoskeletal proteins. These results provide circumstantial evidence that NR2B may play specific roles in function and localization of receptors at excitatory synapses. The possible role of NR2B in early synaptic function gains additional support from functional data suggesting that NMDA receptors have specific roles during development (Komuro and Rakic, 1993; Rabacchi et al., 1992; Yen et al., 1993). The essential role of NR1 and NR2B in development is graphically demonstrated by the neonatal death of transgenic mice lacking either of these two subunits (Forrest et al., 1994; Kutsuwada et al., 1996) whereas NR2A and NR2C-deficient mice are less severely affected (Sakimura et al., 1995; Ebralidze et al., 1996).

Animals↗

PubMed1996

Metabotropic glutamate receptors activate G-protein-coupled inwardly rectifying potassium channels in Xenopus oocytes.

Receptor-mediated activation of a G-protein-coupled inwardly rectifying potassium channel (GIRK) is a common mechanism for synaptic modulation in the CNS. However, evidence for metabotropic glutamate receptor (mGluR) activation of GIRK is virtually nonexistent, despite the widespread and overlapping distribution of these proteins. We examined this apparent paradox by coexpressing mGluRs 1a, 2, and 7 with the GIRK subunits Kir3.1 and Kir3.4 in Xenopus oocytes. Functional expression of GIRK was confirmed by coexpression with the D2 dopamine receptor that is known to activate GIRK in neurons. Agonist activation of each of the three mGluRs evoked inward potassium currents in symmetrical KCI solutions. The current amplitudes evoked by mGluR1a, mGluR2, and D2 were comparable, whereas mGluR7 currents were somewhat smaller. mGluR1a-evoked GIRK currents were not blocked in BAPTA-treated oocytes, demonstrating that GIRK activation was distinct from phospholipase C-mediated activation of the endogenous calcium-dependent chloride current (lCaCl). Pertussis toxin (PTX) treatment significantly reduced both the mGluR and D2 receptor-evoked GIRK currents. In oocytes in which mGluR2 and D2 were coexpressed, activation of mGluR2 occluded additional D2 receptor current, indicating that mGluR2 and D2 receptor coupling to GIRK involves a common G-protein. The efficient coupling of mGluRs to GIRK in oocytes suggests either that mGluR activation of GIRK has been overlooked in neurons or possibly that mGluRs are excluded from GIRK-containing microdomains.

Animals↗

PubMed1996

The impact of receptor desensitization on fast synaptic transmission.

The role of desensitization of ligand-gated channels at fast chemical synapses has been difficult to establish. Densensitization has been studied traditionally with prolonged agonist exposure, whereas the duration of free neurotransmitter in the synaptic cleft is relatively brief. Studies of acetylcholine-, glutamate- and GABA-gated channels using rapid agonist application now provide a means to assess the effects of densensitization in shaping synaptic responses and in influencing neuronal excitability. These data reveal several strikingly different patterns by which the receptor-specific kinetics of densensitization can determine the size, timecourse and frequency of transmitted signals. Densensitization is thus a surprisingly versatile mechanism for shaping synaptic transmission.

Animals↗

PubMed1996

Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific.

Intracellular Ca2+ can reversibly reduce the activity of native N-methyl-D-aspartate (NMDA) receptors in hippocampal neurons, a phenomenon termed Ca2+-dependent inactivation. We examined inactivation in heteromeric NMDA receptors expressed in human embryonic kidney (HEK) 293 cells using whole-cell recording. NR1-1a/2A heteromers showed reversible inactivation that was very similar to native NMDA receptors in cultured hippocampal neurons. Inactivation was dependent on the extracellular Ca2+ concentration and the degree of intracellular Ca2+ buffering. In 2 mM extracellular Ca2+, inactivation resulted in a 46.1 +/- 12.6% reduction in the whole-cell current during a 5-sec agonist application. Inactivation of NR1-1a/2A heteromers was unaffected by calcineurin inhibitors, staurosporine, or phalloidin. NR1-1a/2D heteromers also showed a similar degree of inactivation. In contrast, NR1-1a/2B and NR1-1a/2C heteromers showed no significant inactivation. At saturating concentrations of NMDA (1 mM), NR1-1a/2A heteromers also showed Ca- and glycine-independent desensitization, as seen in native hippocampal neurons. Ca(2+)- and glycine-independent desensitization was less pronounced in NR1-1a/2B heteromers and absent in NR1-1a/2C heteromers. Activation of NR1-1a/2C heteromers triggered intracellular Ca2+ transients similar to NR1-1a/2A heteromers as verified by combined Ca2+ imaging and whole-cell recording. Thus differences in Ca2+ permeability were not responsible for the lack of inactivation in NR1-1a/2C heteromers. Our results show that inactivation of recombinant NMDA receptors requires either the NR2A or NR2D subunit, whereas both inactivation and desensitization were absent in NR2C-containing receptors. The gating of inactivating NMDA receptors is more likely to be influenced by ongoing NMDA receptor activity and Ca2+ transients, perhaps consistent with the prominent expression of NR2A in hippocampus and cerebral cortex.

Biopolymers↗

PubMed1995

L-2-amino-3-phosphonopropionic acid competitively antagonizes metabotropic glutamate receptors 1 alpha and 5 in Xenopus oocytes.

The aspartate analog 2-amino-3-phosphonopropionic acid (AP3) antagonizes glutamate-stimulated phosphatidyl inositide hydrolysis in brain slices, but is reportedly weak or ineffective in antagonizing the phosphatidyl inositide-coupled cloned metabotropic glutamate receptors 1 alpha and 5. Thus we examined the pharmacological properties of AP3 on mGlu1 alpha and mGlu5 receptor responses in Xenopus oocytes. DL-AP3 antagonized mGlu1 alpha and mGlu5 responses, but antagonism was overcome at high glutamate concentrations consistent with competitive inhibition (IC50 = 2.1 mM for mGlu1 alpha). Both responses were also inhibited by (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG). We conclude that the available antagonists cannot distinguish between the mGlu1 alpha receptor and mGlu5 receptor, and that antagonism by AP3 may be obscured in the presence of high agonist concentrations or in cells with spare receptors.

Alanine↗

PubMed1995

Distribution of metabotropic glutamate receptor 7 messenger RNA in the developing and adult rat brain.

The large number of metabotropic glutamate receptor subtypes suggests diverse roles in brain function, although specific distribution patterns can give clues to subtype-specific functions [Hayashi Y. et al. (1993) Nature 366, 687-690; Nakajima Y. et al. (1993) J. biol. Chem. 268, 11868-11873; Nomura A. et al. (1994) Cell 77, 361-369; Ohishi H. et al. (1993), 1009-1018]. The metabotropic glutamate receptor mGluR7 is sensitive to the agonist L-2-amino-4-phosphonobutyric acid, a presynaptic inhibitor of neurotransmitter release. We examined the anatomic distribution of mGluR7 messenger RNA expression by in situ hybridization in the developing and adult rat central nervous systems. Our results demonstrate that mGluR7 messenger RNA is among the most widely distributed of metabotropic glutamate receptors in both the developing and adult rat nervous system and that mGluR7 messenger RNA is expressed in most neuronal groups known to respond to L-2-amino-4-phosphonobutyric acid, including mitral cells of the olfactory bulb, granule cells of the dentate gyrus and neurons of the entorhinal cortex and dorsal root ganglion. mGluR7 exhibits preferential expression in sensory afferent pathways and is highly represented in the periventricular zone of the hypothalamus, the latter implying a modulatory role for mGluR7 in neuroendocrine pathways. Most strikingly, the majority of neurons at all levels of olfactory circuitry are among the areas of highest mGluR7 messenger RNA content. The anatomic distribution of mGluyR7 messenger RNA suggests that mGluR7 activation may participate in the processing of hippocampal, sensory and olfactory information.

Aging↗

PubMed1995

Desensitized states prolong GABAA channel responses to brief agonist pulses.

We studied the role of desensitization at inhibitory synapses by comparing nonequilibrium GABAA channel gating with inhibitory postsynaptic currents (IPSCs). Currents activated by brief pulses of 1-10 mM GABA to outside-out patches from cultured hippocampal neurons mimicked GABA-mediated IPSCs. Although the average open time of single GABAA channels following brief pulses was less than 10 ms, channels entered long (tau = 38-69 ms) closed states and subsequently reopened. Movement through these states resulted in paired-pulse desensitization. The time required for deactivation after removal of agonist also increased in proportion to the extent of desensitization. These results suggest that visits to desensitized states buffer the channel in bound conformations and underlie the expression of long-lasting components of the IPSC. Reopening after GABAA receptor desensitization may thus enhance inhibitory synaptic transmission by prolonging the response to a brief synaptic GABA transient.

Animals↗

PubMed1995

Calcium-dependent inactivation of synaptic NMDA receptors in hippocampal neurons.

1. We examined whether synaptically activated N-methyl-D-aspartate (NMDA) receptors are regulated by intracellular calcium in cultured hippocampal neurons by comparing excitatory postsynaptic currents (EPSCs) to the previously described calcium-dependent regulation of whole cell NMDA currents. Standard whole cell recording and fast application methods were used. 2. Low-frequency (0.2 Hz) stimulation of EPSCs in the presence of 2-amino-5-phosphonovalerate (AP5) evoked a constant amplitude alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated EPSC. On removal of AP5 in Ca(2+)-containing solutions, the amplitude of the slow NMDA receptor-mediated EPSC decreased by approximately 50% during the next 10 stimuli. The decrease in the EPSC was dependent on the extracellular calcium concentration and stimulus frequency, consistent with Ca(2+)-dependent desensitization/inactivation of postsynaptic NMDA receptors. A whole cell prepulse of NMDA (10 microM, 10 s) in Ca(2+)-containing solutions inhibited the slow EPSC to a similar degree. A series of slow EPSCs also produced Ca(2+)-dependent inactivation of whole cell NMDA currents evoked in low calcium solutions. 3. These results demonstrate that synaptic NMDA receptors are inactivated by intracellular calcium and that calcium entry through synaptically activated NMDA receptors is sufficient to provide feedback inhibition of the slow EPSC.

Animals↗

PubMed1995

Synaptic NMDA receptor channels have a low open probability.

Realistic estimates of channel-gating parameters of synaptic receptors are essential to an understanding of synaptic transmission and modulation. However, the gating of N-methyl-D-aspartate (NMDA) channels appears to differ, depending on recording conditions; thus, it remains unclear what measurements are most relevant to synaptic receptors. To further explore this discrepancy, we examined the open probability (Po) of NMDA channels in whole-cell and outside-out patch recording from cultured hippocampal neurons. Currents were evoked by rapid application of saturating concentrations of NMDA in the presence or absence of the "irreversible" open channel blocker, MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) . The reduction of the peak amplitude and the acceleration of the decay of the current in MK-801 were used to derive Po by fitting the current traces to a multistate kinetic model. The Po in whole cell was low (0.04), similar to that previously measured for synaptically activated NMDA channels. In contrast, ensemble average currents from outside-out patches were much more rapidly blocked in the presence of MK-801, indicative of a significantly higher Po. The Po also gradually increased with the duration of recording in both whole-cell and outside-out configurations, suggesting that channel gating is sensitive to mechanical alterations of the patch or that washout of cytoplasmic factors leads to an increase in channel open probability.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons.

Phosphorylation of molecules involved in synaptic transmission by multifunctional protein kinases modulates both pre- and post-synaptic events in the central nervous system. The positioning of kinases near their substrates may be an important part of the regulatory mechanism. The A-kinase-anchoring proteins (AKAPs; ref. 3) are known to bind the regulatory subunit of cyclic AMP-dependent protein kinase A with nanomolar affinity. Here we show that anchoring of protein kinase A by AKAPs is required for the modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate channels. Intracellular perfusion of cultured hippocampal neurons with peptides derived from the conserved kinase binding region of AKAPs prevented the protein kinase A-mediated regulation of AMPA/kainate currents as well as fast excitatory synaptic currents. This effect could be overcome by adding the purified catalytic subunit of protein kinase. A control peptide lacking kinase-binding activity had no effect. To our knowledge, these results provide the first evidence that anchoring of protein kinase A is crucial in the regulation of synaptic function.

A Kinase Anchor Proteins↗

PubMed1994

Glutamate receptor update.

The past year has seen significant advances in matching the actions of recombinant glutamate receptors with the actions of native receptors, and in mapping their distribution and regulation. The discovery of a novel RNA editing mechanism for AMPA receptors and a revised view of the transmembrane topology of the NMDA receptor subunit, NR1, are particularly noteworthy. Seven metabotropic glutamate receptor subtypes have been identified with several interesting expression patterns and transduction mechanisms; results from work on these subtypes has led to a provocative model of the ligand-binding site. Functional studies of metabotropic receptors have been enhanced by the development of the first subtype-specific antagonist.

Animals↗

PubMed1994

Kinetics of AP5 dissociation from NMDA receptors: evidence for two identical cooperative binding sites.

1. N-methyl-D-aspartate (NMDA) channels have two agonist binding sites that have similar binding rates for glutamate. However, it is not known whether the dissociation rates at these two sites, and hence their affinities, are similar. The competitive antagonist, D-2-amino-5-phosphonopentanoic acid (AP5), was used to study dissociation kinetics from NMDA receptors in outside-out patches from cultured hippocampal neurons. 2. Rapid steps from AP5 into NMDA produced currents with a sigmoidal activation time course that was limited by AP5 dissociation. Ensemble average currents were well fitted using kinetic models with two identical, cooperative antagonist binding sites per channel. The results suggest that the two NMDA binding sites have similar affinities, but that occupation of one site reduces the affinity of the other. 3. The agonist and antagonist binding kinetics are consistent with an approximately homogeneous population of NMDA channels in cultured hippocampal neurons.

2-Amino-5-phosphonovalerate↗