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M L Mayer

Publications and source records attributed to M L Mayer.

At least 19 recordsLinked to original sources
PubMed1996

Negative allosteric modulation of wild-type and mutant AMPA receptors by GYKI 53655.

Benzothiadiazides such as cyclothiazide potentiate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor responses, whereas 2,3-benzodiazepines such as 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4 -dihydro- 5H-2,3-benzodiazepine (GYKI 53655) act as noncompetitive antagonists; both drugs act through allosteric modulation. Controversy exists as to whether cyclothiazide and GYKI 53655 act at a common site. Recent mutational analysis has led to the identification of a serine residue in flip splice variants that is critical for directing the interaction of cyclothiazide with AMPA receptors. We tested whether the mutation of this residue to glutamine, which abolishes potentiation by cyclothiazide, can in addition block antagonism by 2,3-benzodiazepines, as would be predicted for action at a common site. We found that the S to Q mutation does not alter antagonism by 2,3-benzodiazepines, suggesting that the molecular determinants directing the interaction between GYKI 53655 and AMPA receptors are not identical to those controlling sensitivity to cyclothiazide. Additional support for this was obtained from analysis of the responses of AMPA receptor flip/flop splice variants, which, despite differences in equilibrium desensitization and sensitivity to cyclothiazide, show only small differences in sensitivity to 2,3-benzodiazepines. Furthermore, introduction of the flip exon from GluRA into GluR6, conferred sensitivity to cyclothiazide but did not increase sensitivity to 2,3-benzodiazepines. Of interest, experiments with native AMPA receptors generated from hippocampal and forebrain poly(A)+ mRNA revealed greater sensitivity to 2,3-benzodiazepines than receptors generated by expression of recombinant AMPA receptors, possibly indicating the existence of an unidentified accessory protein or novel receptor subunit.

Allosteric Regulation↗
PubMed1995

Trapping of glutamate and glycine during open channel block of rat hippocampal neuron NMDA receptors by 9-aminoacridine.

1. N-methyl-D-aspartate (NMDA) receptor responses were recorded from rat hippocampal neurons grown in dissociated culture, using whole-cell, outside-out and nucleated patch recording techniques. Rapid perfusion was used to study voltage-dependent block of NMDA receptors by 9-aminoacridine (9-AA) and by Mg2+. 2. Large amplitude tail currents were evoked on depolarization to +60 mV after application at -100 mV of NMDA and 9-AA but not NMDA and Mg2+. These tail currents were resistant to block by competitive antagonists to the glutamate and glycine binding sites on NMDA receptors and were not evoked when either NMDA or 9-AA were applied alone. 3. The decay kinetics of the tail current were dependent on agonist affinity; the time required for 80% charge transfer was 10-fold briefer for NMDA than for glutamate and 7-fold briefer for L-alanine than for glycine. These results are in accord with a sequential model for block of NMDA receptors by 9-AA, in which neither glutamate nor glycine can dissociate from the open-blocked state of the receptor. 4. Tail current responses had amplitudes 2- to 4-fold larger than responses to maximally effective concentrations of glutamate and glycine, indicating that NMDA receptor channels accumulate in the open-blocked state during co-application of agonist and 9-AA. The rise time and decay kinetics of tail current responses were faster than the response to brief applications of a maximally effective concentration of glutamate. Together, these results suggest that at +60 mV recovery from block by 9-AA occurs faster than the rate of opening of NMDA receptors in response to glutamate. 5. Our experiments suggest that open channel block of NMDA receptors can provide a novel approach for measurement of both open probability and the first latency distribution for ion channel opening in response to the binding of agonists, and provide additional evidence suggesting that the delayed opening of NMDA receptor channels underlies slow activation and deactivation of responses to glutamate.

Aminacrine↗
PubMed1995

Inward rectification of both AMPA and kainate subtype glutamate receptors generated by polyamine-mediated ion channel block.

CA2+-permeable glutamate receptors assembled from subunits containing a GLN residue at the RNA editing site in membrane domain 2 show strong inward rectification. In HEK 293 cells transfected with the kainate receptor subunit GluR6(Q), inward rectification is lost in outside-out patches, suggesting a role for diffusible, cytoplasmic factors. Inclusion of different polyamines in the internal solution restored inward rectification, whereas Mg2+ (1 mM) was inactive. Spermidine (Kd[0 mV] = 5.5 microM) was of higher affinity than spermidine (Kd[0 mV] = 25.4 microM) or putrescine (Kd[0 mV] = 1.2 mM). AMPA receptors assembled from GluRA(flip) showed even higher affinity for spermine (Kd[0 mV] = 1.5 microM). Analysis of the voltage dependence of whole-cell responses predicted intracellular free spermine and spermidine concentrations of 51 and 153 muM, respectively.

Calcium↗
PubMed1995

Structural determinants of allosteric regulation in alternatively spliced AMPA receptors.

The flip and flop splice variants of AMPA receptors show strikingly different sensitivity to allosteric regulation by cyclothiazide; heteromers assembled from GluR-A and GluR-B also exhibit splice variant-dependent differences in efficacy for activation by glutamate and kainate. The sensitivity for attenuation of desensitization by cyclothiazide for homomeric GluR-A was solely dependent upon exchange of Ser-750 (flip) and Asn-750 (flop), and was unaffected by mutagenesis of other divergent residues. In contrast, substantial alteration of the relative efficacy of glutamate versus kainate required mutation of multiple residues in the flip/flop region. Modulation by cyclothiazide was abolished by mutation of Ser-750 to Gin, the residue found at the homologous site in kainate-preferring subunits, whereas introduction of Ser at this site in GluR6 imparted sensitivity to cyclothiazide.

Allosteric Regulation↗
PubMed1995

Structure and function of glutamate and nicotinic acetylcholine receptors.

The past year has seen remarkable progress in defining the structure of various ligand-gated ion channels. Images of opened and closed nicotinic acetylcholine receptors at 9 A resolution have now made it easier to identify the conformational changes underlying gating. In addition, recent studies on glutamate receptors have led to a radical revision of their postulated transmembrane topology: models for agonist-binding and allosteric domains now use sites previously thought to lie in cytoplasmic loops. Other areas that are being actively pursued include identification of the amino acids lining the ion channels, accurate measurements of Ca2+ fluxes, and tests of transmembrane topology in kainate receptor subunits.

Animals↗
PubMed1994

Excitatory amino acid receptors in glial progenitor cells: molecular and functional properties.

We have analyzed the molecular and biophysical properties of glutamate-gated channels in cells of the oligodendrocyte lineage, using both the CG-4 primary cell line (Louis et al: J. Neurosci. Res. 31:193-204, 1992a) and oligodendrocyte progenitors purified from the rat cerebral cortex. CG-4 progenitor cells, as well as primary progenitors, were stained with a specific anti-GABA antibody. In whole-cell patch-clamp recordings, rapid perfusion of the agonists L-glutamate, kainate, and AMPA produced rapidly desensitizing currents in CG-4 cells. NMDA was ineffective. Both rapidly desensitizing and steady-state components of responses to kainate were inhibited by the kainate/AMPA receptor antagonist CNQX. Northern blot analysis of total mRNA isolated from CG-4 cells revealed co-expression of both AMPA- and kainate-preferring glutamate receptor subunits. The activation of glutamate receptors in CG-4 cells caused a rapid and transient elevation of mRNAs for the immediate early gene NGFI-A.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1994

Glial cells of the oligodendrocyte lineage express both kainate- and AMPA-preferring subtypes of glutamate receptor.

mRNAs for AMPA- and kainate-preferring glutamate receptor subunits are expressed abundantly in the CNS, yet functional studies of neurons and glia from brain suggest selective expression of AMPA receptors. We now show that glial cells of the O-2A lineage express rapidly desensitizing responses to kainate, mRNAs for GluR6, GluR7, KA-1, and KA-2, rapidly desensitizing responses to AMPA, and mRNAs for GluR-B, -C, and -D. Analysis of glutamate receptor currents in single cells reveals two receptor populations with high and low affinity for kainate and different sensitivity for potentiation by concanavalin A and for block of desensitization by cyclothiazide. Our experiments describe the characterization of native kainate-preferring receptors in glia and reveal coexpression in single cells of functional AMPA- and kainate-preferring receptors.

Animals↗
PubMed1994

Willardiines differentiate agonist binding sites for kainate- versus AMPA-preferring glutamate receptors in DRG and hippocampal neurons.

Concentration jump responses to 5-substituted (S)-willardiines were recorded from dorsal root ganglion (DRG) and hippocampal neurons under voltage clamp. After block of desensitization by concanavalin-A, dose-response analysis for activation of kainate-preferring receptors in DRG neurons gave the potency sequence trifluoromethyl > iodo > bromo approximately chloro > nitro approximately cyano > kainate > methyl > fluoro > (R,S)-AMPA >> willardiine; EC50 values for the most and least potent willardiine derivatives, 5-trifluoromethyl (70 nM) and 5-fluoro (69 microM), differed 1000-fold. The potency sequence for equilibrium responses at AMPA-preferring receptors in hippocampal neurons was strikingly different from that obtained in DRG neurons: fluoro > cyano approximately trifluoromethyl approximately nitro > chloro approximately bromo > (R,S)-AMPA > iodo > willardiine > kainate > methyl. In hippocampal neurons EC50 values for the most and least potent willardiine derivatives, 5-fluoro (1.5 microM) and 5-methyl (251 microM), differed only 170-fold. Consistent with equilibrium potency measurements, in DRG neurons the kinetics of deactivation for willardiines, recorded following a return to agonist-free solution, were rapid for 5-fluoro (tau off = 43 msec) but slow for 5-iodo (tau off = 4.2 sec), while the opposite sequence was observed for hippocampal neurons, slow for 5-fluoro (tau off = 2.1 sec) and rapid for 5-iodo (tau off = 188 msec). The kinetics of recovery from desensitization showed comparable agonist- and cell-dependent differences. Structure-activity analysis for agonist responses recorded from DRG and hippocampal neurons suggests that for both kainate-preferring and AMPA-preferring receptors the binding of willardiines involves interactions with polar groups such that potency is related to ionization of the uracil ring, and hence the electron-withdrawing ability of the 5-position substituent. However, kainate-preferring receptors differ from AMPA-preferring receptors in possessing a lipophilic pocket that further enhances agonist potency by hydrophobic bonding of the 5-substituent. In contrast, AMPA-preferring receptors lack such a lipophilic site, and for 5-position substituents of the same electron-withdrawing ability, potency decreases with increase in size.

Alanine↗
PubMed1994

Cyclothiazide differentially modulates desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor splice variants.

Agonist responses for flip splice variants of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR-A, -C, and -D are more strongly potentiated by cyclothiazide than are those for the flop forms. Cyclothiazide shows both greater efficacy and higher apparent affinity for potentiation of GluR-Aflip versus GluR-Aflop. Consistent with higher affinity for the flip splice variant, recovery from potentiation by cyclothiazide proceeds 30 times more slowly for GluR-Aflip than for GluR-Aflop. In the presence of 300 microM cyclothiazide a 6-fold leftward shift in the kainate dose-response curve for GluR-Aflip but not GluR-Aflop additionally contributes to a difference in potentiation for these splice variants. Although control responses to glutamate show strong desensitization for both splice variants of GluR-A, in the presence of 100 microM cyclothiazide desensitization is strongly attenuated for GluR-Aflip, whereas for GluR-Aflop desensitization remains pronounced but with a rate of onset slowed 50-fold, compared with control. In heteromeric AMPA receptors formed from GluR-A and GluR-B, the flip splice variants are dominant for controlling both recovery from potentiation of responses to kainate and block of desensitization of responses to glutamate. Our results suggest that the flip/flop module could directly contribute to the binding site for cyclothiazide, raising the possibility that this site is located in an extracellular receptor domain.

Animals↗
PubMed1993

Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin A.

Potentiation by cyclothiazide of recombinant glutamate receptor responses in Xenopus oocytes showed absolute selectivity for AMPA versus kainate receptors. In contrast, concanavalin A strongly potentiated responses at kainate but not AMPA receptors. Rapid desensitization in HEK 293 cells transfected with AMPA receptors was blocked by cyclothiazide, but only weakly attenuated by concanavalin A. Desensitization at kainate receptors was blocked by concanavalin A but unaffected by cyclothiazide. Selective effects of these modulators following coexpression of subunits from different families suggest independent assembly of functional AMPA and kainate receptors. Northern blot analysis of mRNA for dorsal root ganglia revealed a predominant expression of GluR5, indicating that modulation of desensitization by concanavalin A but not cyclothiazide in sensory neurons accurately predicts subunit expression for native glutamate receptors.

Animals↗
PubMed1993

Multiple effects of spermine on N-methyl-D-aspartic acid receptor responses of rat cultured hippocampal neurones.

1. The modulation by polyamines of responses to N-methyl-D-aspartic acid (NMDA) was studied using a rapid perfusion system and whole-cell voltage-clamp recording from rat hippocampal neurons in dissociated culture. 2. Concentration jump responses to 100 microM NMDA in the presence of 10 microM glycine revealed potentiation by 3 mM spermine at a membrane potential of +60 mV, but depression at -120 mV; the degree of potentiation at +60 mV was variable from cell to cell while marked depression at -120 mV was observed in all cells. The depression of responses to NMDA by spermine was highly voltage dependent (z delta = 1.17) with an apparent equilibrium dissociation constant for block at 0 mV of 27 mM. 3. Analysis of spermine dose-potentiation curves for responses recorded at +60 mV in the presence of 10 microM glycine revealed a half-maximal effect at 125 microM. Under the same conditions, but at -60 mV, analysis of spermine-evoked depression was performed for cells with less than 5% potentiation at +60 mV, and revealed half-maximal inhibition at 344 microM. 4. Dose-response analysis for the glycine-sensitive activation of NMDA receptors at +60 mV revealed a 3.5-fold increase in apparent affinity for glycine in the presence of 1 mM spermine. This increase in affinity for glycine was accompanied by a 3.3-fold decrease in the rate of development of glycine-sensitive desensitization, and a 2.4-fold decrease in the rate of dissociation of glycine from NMDA receptors, while the rate constant for dissociation of NMDA was not reduced. 5. In the presence of non-saturating concentrations of glycine, spermine-induced potentiation at +60 mV developed with two exponential components: a slow glycine-sensitive component, the amplitude and time constant of which decreased with increasing glycine concentration (30 nM glycine, amplitude = 80.2 +/- 5.1%, tau = 780 +/- 79 ms; 3 microM glycine, amplitude = 22.6 +/- 7.1%, tau = 45 +/- 13 ms), and a faster component (tau < 20 ms at all concentrations of glycine), the amplitude of which varied from cell to cell, and which became larger with increase in concentration of glycine. When responses to the application of spermine were measured in the presence 10 microM L-alanine instead of 100 nM glycine, the slow component of potentiation was absent.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Hippocampal neurons exhibit cyclothiazide-sensitive rapidly desensitizing responses to kainate.

In whole-cell recordings from mammalian CNS neurons, AMPA-preferring glutamate receptors exhibit strong desensitization in response to AMPA, glutamate, and quisqualate, but not to kainate or domoate. Such desensitization is reduced by lectins, by the nootropic drug aniracetam, and by diazoxide. None of these compounds strongly modulate responses to kainate and domoate, consistent with the apparent lack of desensitization to these agonists. We now report experiments on hippocampal neurons in which responses to kainate were strongly potentiated by cyclothiazide, a benzothiadiazine diuretic and antihypertensive drug structurally related to diazoxide. Cyclothiazide increased the maximum response to a saturating concentration of kainate by approximately 300% and produced a shift to the left in the kainate dose-response curve. Because cyclothiazide was considerably more effective than aniracetam in reducing desensitization evoked by glutamate, we tested the possibility that potentiation of responses to kainate was due to block of a previously undetected component of desensitization in the response to kainate itself. In outside-out patches responses to rapid perfusion of 3 mM kainate showed 34% desensitization, the onset of which developed with a time constant of 2.2 msec. Desensitization of responses to kainate was abolished by 100 microM cyclothiazide, as was the much stronger desensitization evoked by glutamate and AMPA. Cyclothiazide also slowed the rate of deactivation of responses to kainate recorded after return to agonist-free solution. Current-voltage plots for control responses to kainate exhibited outward rectification that was associated with a reduction in the amount of desensitization on depolarization. Both effects were absent in the presence of cyclothiazide, suggesting that rectification of responses to kainate was due to the voltage dependence of desensitization. The complete block of desensitization produced by cyclothiazide provides a powerful new tool for analysis of allosteric regulatory mechanisms at AMPA-preferring glutamate receptors.

Animals↗
PubMed1993

Differential modulation by cyclothiazide and concanavalin A of desensitization at native alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and kainate-preferring glutamate receptors.

Concanavalin A, cyclothiazide, and aniracetam, ligands that modulate desensitization at glutamate receptors, were tested for their actions on responses at kainate-preferring receptors in dorsal root ganglion (DRG) neurons and at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring receptors in hippocampal neurons. In DRG neurons concanavalin A blocked desensitization produced by either kainate or 5-chlorowillardiine and strongly potentiated the peak amplitude of responses to both agonists. However, in hippocampal neurons concanavalin A produced only weak potentiation of responses to kainate and 5-chlorowillardiine, and after treatment with lectin responses to 5-chlorowillardiine remained strongly desensitizing. In contrast, cyclothiazide completely blocked desensitization produced by 5-chlorowillardiine in hippocampal neurons and strongly potentiated responses to kainate; the action of aniracetam was similar but much weaker. In DRG neurons cyclothiazide and aniracetam had no effect on desensitization and instead produced weak inhibition of responses to kainate. The different sensitivities of native AMPA- and kainate-preferring glutamate receptors to cyclothiazide and concanavalin A should prove useful for the differentiation of glutamate receptor subtypes in other areas of the central nervous system.

Alanine↗
PubMed1992

CDC43 and RAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase.

The question regarding the identity of the alpha and beta subunits of the yeast type I protein geranylgeranyltransferase was explored using prokaryotic expression of candidate genes. The Saccharomyces cerevisiae CDC43 and RAM2 genes were expressed in Escherichia coli and cell extracts examined for the ability to transfer [3H]geranylgeranyl diphosphate to an appropriate CaaX protein substrate. Individual expression of each gene yielded no activity; however, co-expression of the two genes resulted in high levels of [3H] geranylgeranyl incorporation into the substrate protein Ras-Cys-Val-Val-Leu. The activity was partially purified yielding approximately 12,600 units/liter. The partially purified enzyme geranylgeranylated the Ras-Cys-Val-Val-Leu, Ras-Cys-Ala-Ile-Leu, Ras-Cys-Ile-Ile-Leu, and Ras-Cys-Thr-Ile-Leu substrates but not the Ras-Cys-Val-Leu-Ser or Ras-Ser-Val-Leu-Ser substrates. The protein geranylgeranyltransferase was highly specific for geranylgeranyl diphosphate and poorly transferred farnesyl. The recombinant enzyme was indistinguishable from the native type I geranylgeranyltransferase in yeast extracts. As has been reported for the protein farnesyltransferase, the yeast type I protein geranylgeranyltransferase is also a magnesium-requiring, zinc metalloenzyme. Interestingly, the recombinant enzyme functioned with calcium as the only divalent cation, although addition of zinc increased calcium-dependent activity 2-fold.

Alkyl and Aryl Transferases↗
PubMed1992

Activation and desensitization of AMPA/kainate receptors by novel derivatives of willardiine.

Willardiine [(S)-1-(2-amino-2-carboxyethyl)pyrimidine-2,4-dione] is a naturally occurring heterocyclic excitatory amino acid present in the seeds of Acacia and Mimosa. A series of 5-substituted willardiines were synthesized in single enantiomeric forms and tested for activity at AMPA/kainate receptors, using whole-cell recording from mouse embryonic hippocampal neurons. The (S)- but not (R)-isomers of willardiine and 5-bromowillardiine were potent agonists, producing rapidly but incompletely desensitizing responses. At equilibrium, (S)-5-fluorowillardiine (EC50, 1.5 microM) was seven times more potent than (R,S)-AMPA (EC50, 11 microM) and 30 times more potent than willardiine (EC50, 45 microM); the potency sequence was fluoro greater than nitro greater than chloro approximately bromo greater than iodo greater than willardiine. Willardiines produce strikingly different degrees of desensitization: at saturating doses the equilibrium response to the weakly desensitizing agonist (S)-5-iodowillardiine was similar in amplitude to the response to kainate and 10 times larger than the response to the strongly desensitizing agonist (S)-willardiine. The desensitization sequence was fluoro greater than willardiine greater than nitro approximately chloro greater than bromo greater than iodo greater than kainate. Cross-desensitization experiments confirm that willardiines bind to the same receptors activated by kainate and AMPA, and show that both the rapidly desensitizing and equilibrium responses to willardiines are mediated by the same receptor: (S)-5-iodowillardiine blocked activation of the rapidly desensitizing response evoked by (S)-willardiine and (S)-5-fluorowillardiine, while the latter agonists blocked the equilibrium response to (S)-5-iodowillardiine. A slowly decaying inward tail current was recorded after a brief application of (S)-5-fluorowillardiine but not (S)-willardiine, consistent with a model in which willardiines bind with different affinity to desensitized receptors, such that following removal of agonist, receptors trapped in the desensitized state can return to the open state before dissociation of agonist terminates receptor activation. Willardiines are the first compounds characterized in which simple changes in molecular structure are associated with marked differences in the ability of agonists to produce desensitization of AMPA/kainate receptors.

Alanine↗
PubMed1992

Effect of extracellular pH on the potency of N-methyl-D-aspartic acid receptor competitive antagonists.

Structure-activity analysis reveals that acidic alpha-amino acids containing an omega-PO3H2 group are more potent antagonists at N-methyl-D-aspartate (NMDA) receptors than are analogs with omega-COOH or omega-tetrazole groups. At physiological values of extra-cellular pH the omega-PO3H2 group is only partially deprotonated and the corresponding antagonists exist as ions with one or two negative charges. In contrast, competitive antagonists with omega-COOH and omega-tetrazole groups are fully ionized at physiological pH but carry only a single negative charge. Dose-inhibition analysis was performed with (2R)-AP7 and its piperidine derivative LY 257883 to determine whether ionization of the omega-PO3H2 group influences NMDA receptor antagonist potency; these experiments revealed a > 3-fold increase in potency on raising of the extracellular pH from 7.3 to pH 8.2, consistent with the increase in the relative concentration of the ionic form of the antagonist in which the omega-PO3H2 group contains two negative charges. Experiments with the omega-COOH-containing analog of LY 257883 and with SDZ EAB 515, an omega-PO3H2-containing antagonist of novel structure, revealed only 1.5- and 1.3-fold increases in potency, respectively, over the same pH range. Analysis of the kinetics of block of NMDA-activated currents resulting from rapid application of LY 257883 suggests that the increase in potency on raising of the extracellular pH results largely from an increase in the antagonist association rate constant but also from a small decrease in the dissociation rate constant. Together, these results suggest that the fully ionized forms of the R-enantiomers of AP7 and LY 257883 act as the active antagonist species at NMDA receptors.

2-Amino-5-phosphonovalerate↗