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THE ENDOGENEOUS AGONIST QUINOLINIC ACID AND THE NON ENDOGENOUS HOMOQUINOLINIC ACID DISCRIMINATE BETWEEN NMDAR2 RECEPTOR SUBUNITS

Quinolinic acid is an endogenous neurotoxin with NMDA receptor agonist properties. As such it may be the etiologic agent in many diseases. In this paper the NMDA receptor agonist properties of quinolinic acid, as well as those of homoquinolinic acid, a non endogenous analogue, were investigated in Xenopus oocytes injected with 12-day-old rat cortical mRNA or with recombinant NMDA receptors. In oocytes injected with cortical mRNA, quinolinic acid was a weak NMDA receptor agonist: millimolar concentrations were necessary to induce responses that were smaller than maximal responses induced by NMDA; homoquinolinic acid and NMDA had similar affinities but different efficacies: maximal responses induced by homoquinolinic acid were larger than maximal responses induced by NMDA. Cortical mRNA, as verified by RT-PCR and restriction analysis, contains various NMDA subunits. In order to investigate if the low affinity or efficacy of quinolinic acid could be explained by receptor composition, the pharmacological properties of the putative agonists were investigated in oocytes expressing binary combinations of recombinant NMDA receptors. Quinolinic acid did not activate receptors containing NR1 + NR2C but did activate receptors containing NR1 + NR2A and NR1 + NR2B even if only at millimolar concentrations; homoquinolinic acid activated all subunit combinations but was less efficient than NMDA only in the NR1 + NR2C subunit combination. The relative efficacies of quinolinic acid and homoquinolinic acid were evaluated by comparing the maximal responses induced by these agonists with those induced by NMDA and glutamate in the same oocytes. The rank order of potency was quinolinic acid < NMDA < homoquinolinic acid < or = glutamate for the NR1 + NR2A and NR1 + NR2B combinations whereas for NR1 + NR2C it was quinolinic acid << << homoquinolinic acid < NMDA < or = glutamate. The use of quinolinic acid and homoquinolinic acid may thus help to identify endogenous receptors containing the NR2C subunit.

Implications of the quaternary twist allosteric model for the physiology and pathology of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are pentameric ligand-gated ion channels composed of subunits that consist of an extracellular domain that carries the ligand-binding site and a distinct ion-pore domain. Signal transduction results from the allosteric coupling between the two domains: the distance from the binding site to the gate of the pore domain is 50 Å. Normal mode analysis with a Cα Gaussian network of a new structural model of the neuronal α7 nAChR showed that the lowest mode involves a global quaternary twist motion that opens the ion pore. A molecular probe analysis, in which the network is modified at each individual amino acid residue, demonstrated that the major effect is to change the frequency, but not the form, of the twist mode. The largest effects were observed for the ligand-binding site and the Cys-loop. Most (24/27) of spontaneous mutations known to cause congenital myasthenia and autosomal dominant nocturnal frontal lobe epilepsy are located either at the interface between subunits or, within a given subunit, at the interface between rigid blocks. These interfaces are modified significantly by the twist mode. The present analysis, thus, supports the quaternary twist model of the nAChR allosteric transition and provides a qualitative interpretation of the effect of the mutations responsible for several receptor pathologies.

Convulsions induced by submaximal dose of pentylenetetrazol in mice are antagonized by the benzodiazepine antagonist Ro 15-1788

The effects of benzodiazepine antagonist Ro 15-1788, alone or with diazepam, were studied in mice on convulsions induced by pentylenetetrazol (PTZ). We found that Ro 15-1788 (1 mg/kg) was able to antagonize the anticonvulsive effects of diazepam (1 mg/kg), but also had, with submaximal doses of PTZ (65 mg/kg), its own anticonvulsive action. At very low doses (0.1 mg/kg), it even potentiated the anticonvulsive effects of diazepam (0.05 mg/kg). This dual action provides evidence for partial agonist properties of the antagonist Ro 15-1788.

An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors

The molecular mechanisms of nicotinic receptor activation are still largely unknown. The crystallographic structure of the acetylcholine binding protein (AChBP) reveals a single H‐bond between two different acetylcholine binding loops. Within these homologous loops we systematically introduced α4 residues into the α7/5HT3 chimeric receptor and found that the single point mutations G152K (loop B) and P193I (loop C) displayed a non‐additive increase of equilibrium binding affinity for several agonists compared with the double mutant G152K/P193I. In whole‐cell patch–clamp recordings, G152K, P193I and G152K/P193I mutants displayed an increase up to 5‐fold in acetylcholine potency with a large decrease of the apparent Hill coefficients (significantly smaller than one). Concomitantly, the G152K/P193I mutant showed a dramatic loss of high‐affinity α‐bungarotoxin binding (100‐fold decrease), thus pinpointing a new contact area for the toxin. Fitting the data with an allosteric–kinetic model, together with molecular dynamic simulations, suggests that the presence of the inter‐backbone H‐bond between positions 152 and 193, revealed in α4 and in α7 double mutant but not in α7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.

Characterization of convulsions induced by methyl β-carboline-3-carboxylate in mice

The convulsive properties of methyl beta-carboline-3-carboxylate (beta-CCM) were evaluated in mice. When injected subcutaneously at a dose of 10 mg/kg beta-CCM induced convulsions in 75% of the mice with a median latency of 2.12 +/- 0.25 min. The CD50 was determined to be about 5 mg/kg. Electroencephalographic recordings showed that convulsions were brief (10 s), of cortical origin and propagating rapidly to the hippocampus. EEG alterations induced by low doses of beta-CCM lasted up to 1 h. The convulsive effect of beta-CCM was compared to that of PTZ. PTZ-induced convulsions occurred with a longer latency (9.26 +/- 1.33 min). beta-CCM and PTZ could act synergistically when injected in non-convulsive doses. When beta-CCM was injected 2-30 min before pentylenetetrazol (PTZ) there was a clear potentiation of the convulsive effect of PTZ. The convulsions induced by beta-CCM were blocked by diazepam (DZ) and by Ro 15-1788. In addition, beta-CCM reversed the sedative effect of a high dose of DZ for more than 30 min. Our results confirm that beta-CCM acts through the BZ receptor and indicate that the effects induced by a single dose of beta-CCM last more than 30 min.

Agonist trapped in ATP-binding sites of the P2X2 receptor

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent “tethering” reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.

Demonstration of the partial agonist profiles of Ro 16-6028 and Ro 17-1812 in mice in vivo

Benzodiazepine receptor occupancy by the full agonist, diazepam, and by the two putative partial agonists, Ro 16-6028 and Ro 17-1812, was measured by inhibition of in vivo [3H]Ro 15-1788 binding in mouse brain and was correlated with their pharmacological effects. The anticonvulsant effects of Ro 16-6028, Ro 17-1812 and diazepam (ED50 values) appeared at receptor occupancies of 40, 20 and less than 5%, respectively. Moreover, at the highest measurable receptor occupancy (90-100%), Ro 16-6028 and Ro 17-1812 did not induce any rotarod deficit whereas a complete deficit was observed with diazepam at 35% receptor occupancy.

A Putative Extracellular Salt Bridge at the Subunit Interface Contributes to the Ion Channel Function of the ATP-gated P2X2 Receptor

The recent crystal structure of the ATP-gated P2X4 receptor revealed a static view of its architecture, but the molecular mechanisms underlying the P2X channels activation are still unknown. By using a P2X2 model based on the x-ray structure, we sought salt bridges formed between charged residues located in a region that directly connects putative ATP-binding sites to the ion channel. To reveal their significance for ion channel activation, we made systematic charge exchanges and measured the effects on ATP sensitivity. We found that charge reversals at the interfacial residues Glu63 and Arg274 produced gain-of-function phenotypes that were cancelled upon paired charge swapping. These results suggest that a putative intersubunit salt bridge formed between Glu63 and Arg274 contributes to the ion channel function. Engineered cysteines E63C and R274C formed redox-dependent cross-links in the absence of ATP. By contrast, the presence of ATP reduced the rate of disulfide bond formation, indicating that ATP binding might trigger relative movement of adjacent subunits at the level of Glu63 and Arg274, allowing the transmembrane helices to open the channel.

Electrophysiological and pharmacological properties of GluR1, a subunit of a glutamate receptor-channel expressed in Xenopus oocytes

A cDNA clone encoding an excitatory amino acid receptor was isolated from a rat brain cDNA library by Hollmann et al. (Nature, 342 (1989) 643-648). In Xenopus oocytes, this clone, GluR1, expressed a functional receptor-channel activated by kainate (KA), domoate (D), glutamate and quisqualate (QA). The apparent affinity (EC50) for QA (0.1 microM) was higher than that for KA (50 microM). The maximal response to QA was about 1/10 of that to KA. QA inhibited the KA induced current. The N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3 dione (DNQX) competitively blocked the effects of both agonists. Currents induced by KA, QA and D in oocytes expressing GluR1 showed identical voltage sensitivities. GluR1 and KA receptor-channels expressed from rat striatum poly(A)+ RNA showed the same ionic selectivity, being permeable mostly to Na+ and K+. The current-voltage relationships of GluR1 showed a strong inward rectification, whereas those of KA receptor-channels expressed from poly(A)+ RNA from various rat brain regions were more linear.

Distinct subcellular targeting of fluorescent nicotinic α3β4 and serotoninergic 5-HT3A receptors in hippocampal neurons

The nicotinic acetylcholine receptors (nAChRs) and the 5‐HT3 serotonin receptor subtype belong to a superfamily of neurotransmitter‐gated ion channels involved in fast synaptic communication throughout the nervous system. Their trafficking to the neuron plasmalemma, as well as their targeting to specific subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N‐termini of α3β4‐nAChR subunits and the 5‐HT3AR subunit with cyan and yellow fluorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK‐293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric α3CFP‐β4 and YFP‐α3β4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP‐5‐HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP‐5‐HT3A receptor was found to be targeted to the micropodia in HEK‐293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied specific fluorescent probes. The efficient targeting of the YFP‐5‐HT3A to the cytoplasmic membrane is in line with the large serotonin‐elicited currents (nA range) measured by whole‐cell voltage‐clamp recordings in transfected HEK‐293 cells. In contrast, α3β4‐nAChRs expressed in the same cells yielded weaker ACh‐evoked responses. Taken together, the fluorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of α3β4‐nACh receptors and the predominant expression of the 5‐HT3AR in dendritic surface loci.