Glucagon-like peptide-1 receptor signaling modifies the extent of diabetic kidney disease through dampening the receptor for advanced glycation end products-induced inflammation.

Glucagon like peptide-1 (GLP-1) is a hormone produced and released by cells of the gastrointestinal tract following meal ingestion. GLP-1 receptor agonists (GLP-1RA) exhibit kidney-protective actions through poorly understood mechanisms. Here we interrogated whether the receptor for advanced glycation end products (RAGE) plays a role in mediating the actions of GLP-1 on inflammation and diabetic kidney disease. Mice with deletion of the GLP-1 receptor displayed an abnormal kidney phenotype that was accelerated by diabetes and improved with co-deletion of RAGE in vivo. Activation of the GLP-1 receptor pathway with liraglutide, an anti-diabetic treatment, downregulated kidney RAGE, reduced the expansion of bone marrow myeloid progenitors, promoted M2-like macrophage polarization and lessened markers of kidney damage in diabetic mice. Single cell transcriptomics revealed that liraglutide induced distinct transcriptional changes in kidney endothelial, proximal tubular, podocyte and macrophage cells, which were dominated by pathways involved in nutrient transport and utilization, redox sensing and the resolution of inflammation. The kidney-protective action of liraglutide was corroborated in a non-diabetic model of chronic kidney disease, the subtotal nephrectomised rat. Thus, our findings identify a novel glucose-independent kidney-protective action of GLP-1-based therapies in diabetic kidney disease and provide a valuable resource for exploring the cell-specific kidney transcriptional response ensuing from pharmacological GLP-1R agonism.

Molecular determinants of subtype-selective efficacies of cytisine and the novel compound NS3861 at heteromeric nicotinic acetylcholine receptors.

Deciphering which specific agonist-receptor interactions affect efficacy levels is of high importance, because this will ultimately aid in designing selective drugs. The novel compound NS3861 and cytisine are agonists of nicotinic acetylcholine receptors (nAChRs) and both bind with high affinity to heteromeric α3ß4 and α4ß2 nAChRs. However, initial data revealed that the activation patterns of the two compounds show very distinct maximal efficacy readouts at various heteromeric nAChRs. To investigate the molecular determinants behind these observations, we performed in-depth patch clamp electrophysiological measurements of efficacy levels at heteromeric combinations of α3- and α4-, with ß2- and ß4-subunits, and various chimeric constructs thereof. Compared with cytisine, which selectively activates receptors containing ß4- but not ß2-subunits, NS3861 displays the opposite ß-subunit preference and a complete lack of activation at α4-containing receptors. The maximal efficacy of NS3861 appeared solely dependent on the nature of the ligand-binding domain, whereas efficacy of cytisine was additionally affected by the nature of the ß-subunit transmembrane domain. Molecular docking to nAChR subtype homology models suggests agonist specific interactions to two different residues on the complementary subunits as responsible for the ß-subunit preference of both compounds. Furthermore, a principal subunit serine to threonine substitution may explain the lack of NS3861 activation at α4-containing receptors. In conclusion, our results are consistent with a hypothesis where agonist interactions with the principal subunit (α) primarily determine binding affinity, whereas interactions with key amino acids at the complementary subunit (ß) affect agonist efficacy.

In vitro pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107.

Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity alpha7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

Molecular imaging of alpha7 nicotinic acetylcholine receptors: design and evaluation of the potent radioligand [18F]NS10743.

PURPOSE: The outstanding diversity of cellular properties mediated by neuronal and nonneuronal alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) points to the diagnostic potential of quantitative nuclear molecular imaging of alpha7 nAChR in neurology and oncology. It was our goal to radiolabel the alpha7 nAChR agonist 4-[5-(4-fluoro-phenyl)-[1,3,4]oxadiazol-2-yl]-1,4-diaza-bicyclo[3.2.2]nonane (NS10743) and to assess the selectivity of [(18)F]NS10743 binding site occupancy in animal experiments. METHODS: [(18)F]NS10743 was synthesized by nucleophilic substitution of the nitro precursor. In vitro receptor affinity and selectivity were assessed by radioligand competition and autoradiography. The radiotracer properties were evaluated in female CD-1 mice by brain autoradiography and organ distribution. Target specificity was validated after treatment with SSR180711 (10 mg/kg, intraperitoneal), and metabolic stability was investigated using radio-HPLC. RESULTS: The specific activity of [(18)F]NS10743 exceeded 150 GBq/micromol at a radiochemical purity >99%. In vitro, NS10743 and [(18)F]NS10743 showed high affinity and specificity towards alpha7 nAChR. The brain permeation of [(18)F]NS10743 was fast and sufficient with values of 4.83 and 1.60% injected dose per gram and brain to plasma ratios of 3.83 and 2.05 at 5 and 60 min after radiotracer administration. Brain autoradiography and organ distribution showed target-specific accumulation of [(18)F]NS10743 in brain substructures and various alpha7 nAChR-expressing organs. The radiotracer showed a high metabolic stability in vivo with a single polar radiometabolite, which did not cross the blood-brain barrier. CONCLUSION: The good in vitro and in vivo features of [(18)F]NS10743 make this radioligand a promising candidate for quantitative in vivo imaging of alpha7 nAChR expression and encourage further investigations.

Broad-spectrum efficacy across cognitive domains by alpha7 nicotinic acetylcholine receptor agonism correlates with activation of ERK1/2 and CREB phosphorylation pathways.

The alpha7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel alpha7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (Ki = 10.8 nM) and human (Ki = 16.7 nM) alpha7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the alpha7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that alpha7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of alpha7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked alpha7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that alpha7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimer's disease and schizophrenia.

The alpha7 nicotinic receptor agonist SSR180711 increases activity regulated cytoskeleton protein (Arc) gene expression in the prefrontal cortex of the rat.

Nicotinic alpha7 acetylcholine receptors (alpha7 nAChR) have been shown to enhance attentional function and aspects of memory function in experimental models and in man. The protein Arc encoded by the effector immediate early gene arc or arg3.1 has been shown to be strongly implicated in long-term memory function. We have sought to determine if alpha7 nAChR mediate the stimulation of arc gene expression, and if so, where in the brain such activation may occur using semi-quantitative in situ hybridisation. Administration of the novel and selective alpha7 nAChR agonist, SSR180711 (1, 3 and 10 mg/kg) to adolescent rats, produced a dose- and time-dependent increase in the expression of Arc mRNA in the prefrontal cortex and the ventral orbital cortex. By contrast, no change in mRNA levels was detected in the parietal cortex and the CA1 of the hippocampus. These data show that alpha7 nAChR activates a subset of neurons in the rat prefrontal cortex and this activation likely is important for the attentional effects of this new class of drugs.

Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells.

Nicotinic acetylcholine receptors (nAChR's) containing the α6 subunit (α6) are putative drug targets of relevance to Parkinson's disease and nicotine addiction. However, heterologous expression of α6 receptors has proven challenging which has stifled drug discovery efforts. Here, we investigate potential new avenues for achieving functional α6 receptor expression. Combinations of chimeric and mutated α6, ß2 and ß3 subunits were co-expressed in the human HEK293 cell line and receptor expression was assessed using Ca(2+)-imaging (FLIPR™) and whole-cell patch-clamp electrophysiology. Transient transfections of a chimeric α6/α3 subunit construct in combination with ß2 and ß3(V9'S) gave rise to significant acetylcholine-evoked whole-cell currents. Increasing the ß3(V9'S):ß2:α6/α3 cDNA ratio, resulted in a significantly higher fraction of cells with robust current levels. Using an excess of wild-type ß3, significant functional expression of α6/α3ß2ß3 was also demonstrated. Comparing the acetylcholine concentration-response relationship of α6/α3ß2ß3(V9'S) to that of α6/α3ß2ß3 revealed the ß3 point mutation to result in decreased current decay rate and increased ACh agonist potency. Ca(2+)-imaging experiments showed preservation of basic α6 receptor pharmacology. Our results establish that α6/α3ß2ß3(V9'S) replicate several basic features of native α6 receptors but also highlight several caveats associated with using this construct and may therefore provide guidance for future drug hunting efforts.

11C-NS14492 as a novel PET radioligand for imaging cerebral alpha7 nicotinic acetylcholine receptors: in vivo evaluation and drug occupancy measurements.

UNLABELLED: Small-molecule α(7) nicotinic acetylcholine receptor (α(7)nAChR) agonists are currently validated for use as treatment for cognitive disturbances in schizophrenia and in Alzheimer disease. A suitable radiolabeled α(7)nAChR PET tracer would be important for in vivo quantification of α(7)nAChR binding in humans and to measure α(7)nAChR occupancy of α(7)nAChR drug candidates. Here, we present the radiosynthesis and in vivo evaluation of (11)C-NS14492 as a selective α(7)nAChR PET radioligand. METHODS: The high-affinity α(7)nAChR-selective partial agonist NS14492 was radiolabeled by methylation of its desmethyl precursor using (11)C-methyl triflate. Female Danish Landrace pigs were studied at baseline and after intravenous administration of blocking doses of either the α(7)nAChR partial agonist SSR180711 or the unlabeled NS14492. (11)C-NS14492 was given as an intravenous bolus injection, and the pigs were scanned for 90 min both at baseline and in the blocked conditions. Arterial blood was collected during the scanning, plasma was counted, and parent compound fraction was determined with radio-high-performance liquid chromatography. PET data were quantified with a graphical analysis with arterial input; (11)C-NS14492 regional distribution volumes were calculated, and α(7)nAChR occupancy was determined using an occupancy plot. RESULTS: (11)C-NS14492 had a high uptake in the pig brain, with the highest binding in the cerebral cortex and thalamus in accordance with α(7)nAChR distribution. Pretreatment with NS14492 and SSR180711 consistently decreased distribution volumes of (11)C-NS14492 in all examined regions, in a dose-dependent manner, supporting the finding that the radioligand binds selectively to α(7)nAChR in vivo. CONCLUSION: We report here that (11)C-NS14492 is the first, to our knowledge, PET radioligand for α(7)nAChR showing a dose-dependent decline in cerebral binding after receptor blockade. This compound is considered a promising PET tracer for in vivo measurements of α(7)nAChR binding in the human brain.

Distinct structural features of cyclothiazide are responsible for effects on peak current amplitude and desensitization kinetics at iGluR2.

Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission. Upon glutamate application, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptors undergo rapid and almost complete desensitization that can be attenuated by positive allosteric modulators. The molecular mechanism of positive allosteric modulation has been elucidated previously by crystal structures of the ligand-binding core of iGluR2 in complex with, for example, cyclothiazide (CTZ). Here, we investigate the structure and function of CTZ and three closely related analogues NS1493, NS5206, and NS5217 at iGluR2, by X-ray crystallography and fast application patch-clamp electrophysiology. CTZ was the most efficacious and potent modulator of the four compounds on iGluR2(Q)(i) [E(max) normalized to response of glutamate: 754% (CTZ), 490% (NS1493), 399% (NS5206), and 476% (NS5217) and EC(50) in micromolar: 10 (CTZ), 26 (NS1493), 43 (NS5206), and 48 (NS5217)]. The four modulators divide into three groups according to efficacy and desensitization kinetics: (1) CTZ increases the peak current efficacy twice as much as the three analogues and nearly completely blocks receptor desensitization; (2) NS5206 and NS5217 have low efficacy and only attenuate desensitization partially; (3) NS1493 has low efficacy but nearly completely blocks receptor desensitization. A hydrophobic substituent at the 3-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system is important for compound efficacy, with the following ranking: norbornenyl (bicyclo[2.2.1]hept-2-ene)>cyclopentyl>methyl. The replacement of the norbornenyl moiety with a significantly less hydrophobic cyclopentane ring increases the flexibility of the modulator as the cyclopentane ring adopts various conformations at the iGluR2 allosteric binding site. The main structural feature responsible for a nearly complete block of desensitization is the presence of an NH hydrogen bond donor in the 4-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system, forming an anchoring hydrogen bond to Ser754. Therefore, the atom at the 4-position of CTZ seems to be a major determinant of receptor desensitization kinetics.

Alpha7 nicotinic acetylcholine receptor activation ameliorates scopolamine-induced behavioural changes in a modified continuous Y-maze task in mice.

The alpha7 (alpha7) nicotinic acetylcholine receptor may represent a drug target for the treatment of disorders associated with working memory/attentional dysfunction. We investigated the effects of three distinct alpha7 nicotinic acetylcholine receptor agonists: 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941; 0.01-0.1 mg/kg), 4-bromophenyl 1,4-diazabicyclo(3.2.2) nonane-4-carboxylate (SSR180711; 0.3-3 mg/kg) and N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide (PNU-282987; 1-10 mg/kg), on scopolamine-induced deficits in a modified Y-maze procedure. Mice were forced to choose one of two visually distinct arms, and were confined there for a 5 min exploration period before being allowed to explore both arms for a 2 min test session, immediately thereafter. The time spent in each arm, entries and total distance travelled were recorded using an automated system. Characterisation experiments showed that scopolamine-treated (1 mg/kg) mice spent less time exploring the unfamiliar arm, when compared with vehicle-treated animals. Combination experiments showed that all three alpha7 agonists ameliorated scopolamine-induced changes in unfamiliar arm exploration. In conclusion, the present data support the idea that alpha7 nicotinic acetylcholine receptors may represent an interesting target for the treatment of conditions associated with attentional/working memory dysfunction.

Evaluation of alpha7 nicotinic acetylcholine receptor agonists and positive allosteric modulators using the parallel oocyte electrophysiology test station.

Neuronal acetylcholine receptors (nAChRs) of the alpha7 subtype are ligand-gated ion channels that are widely distributed throughout the central nervous system and considered as attractive targets for the treatment of various neuropsychiatric and neurodegenerative diseases. Both agonists and positive allosteric modulators (PAMs) are being developed as means to enhance the function of alpha7 nAChRs. The in vitro characterization of alpha7 ligands, including agonists and PAMs, relies on multiple technologies, but only electrophysiological measurements assess the channel activity directly. Traditional electrophysiological approaches utilizing two-electrode voltage clamp or patch clamp in isolated cells have very low throughput to significantly impact drug discovery. Abbott (Abbott Park, IL) has developed a two-electrode voltage clamp-based system, the Parallel Oocyte Electrophysiology Test Station (POETs()), that allows for the investigation of ligand-gated ion channels such as alpha7 nAChRs in a higher-throughput manner. We describe the utility of this technology in the discovery of selective alpha7 agonists and PAMs. With alpha7 agonists, POETs experiments involved both single- and multiple-point concentration-response testing revealing diverse activation profiles (zero efficacy desensitizing, partial, and full agonists). In the characterization of alpha7 PAMs, POETs testing has served as a reliable primary or secondary screen identifying compounds that fall into distinct functional types depending on the manner in which current potentiation occurred. Type I PAMs (eg, genistein, NS1738, and 5-hydroxyindole) increase predominantly the peak amplitude response, type II PAMs affect the peak current and current decay (eg, PNU-120,596 and 4-(naphthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), and anothertype slowing the current decay kinetics in the absence of increases in the peak current. In summary, POETs technology allows for significant impact on higher throughput in the testing of alpha7 agonists and PAMs and for identification of compounds with unique profiles that could prove valuable in identifying an optimum in vitro profile in the development of therapeutics for which the alpha7 subtype is considered.

Role of channel activation in cognitive enhancement mediated by alpha7 nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: Several agonists of the alpha7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid alpha7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved. EXPERIMENTAL APPROACH: Two structurally related alpha7 nAChR agonists were characterized and used to assess the degree of efficacy required in a behavioural paradigm. KEY RESULTS: NS6784 activated human and rat alpha7 nAChR with EC(50)s of 0.72 and 0.88 microM, and apparent efficacies of 77 and 97% respectively. NS6740, in contrast, displayed little efficacy at alpha7 nAChR (<2% in oocytes, < or =8% in GH4C1 cells), although its agonist-like properties were revealed by adding a positive allosteric modulator of alpha7 nAChRs or using the slowly desensitizing alpha7V274T receptor. In mouse inhibitory avoidance (IA) memory retention, NS6784 enhanced performance as did the 60% partial agonist A-582941. In contrast, NS6740 did not enhance performance, but blocked effects of A-582941. CONCLUSIONS AND IMPLICATIONS: Collectively, these findings suggest that a degree of alpha7 nAChR agonist efficacy is required for behavioural effects in the IA paradigm, and that such behavioural efficacy is not due to alpha7 nAChR desensitization. Also, a partial agonist of very low efficacy for this receptor could be used as an inhibitor, in the absence of alpha7 nAChR antagonists with favourable CNS penetration.

Alpha-7 nicotinic acetylcholine receptor agonists selectively activate limbic regions of the rat forebrain: an effect similar to antipsychotics.

It is considered that activation of nicotinic alpha7 receptors (alpha7 nAChR) is useful for the treatment of cognitive disturbances in schizophrenia and Alzheimer's disease. Recently, selective alpha7 nAChR agonists have been discovered and are used to validate the alpha7 nAChR as a drug target for the treatment of cognitive disturbances in schizophrenia. One important feature shared by all known antipsychotics is their capacity to induce expression of the neuronal immediate-early gene c-fos in the limbic forebrain. Using two novel and selective alpha7 nAChR agonists, PNU-282987 and SSR180711, we investigated their ability to induce c-Fos expression in the limbic forebrain with particular emphasis on the same regions reported to be activated by antipsychotics. Both alpha7 nAChR agonists increased c-Fos dose-dependently in the prefrontal cortex and the shell of nucleus accumbens, while leaving the core of nucleus accumbens and the dorsolateral striatum unaffected. The accumbal and cortical effect of SSR180711 was blocked completely by pre-administration of the alpha7 nAChR antagonist methyllycaconitine. Also, SSR180711 displayed no c-Fos-inducing effect in alpha7 nAChR knock-out mice. In conclusion, these results show that selective pharmacologic stimulation of alpha7 nAChR function results in activation of forebrain regions similar to conventional antipsychotics.

Partial agonism and antagonism of the ionotropic glutamate receptor iGLuR5: structures of the ligand-binding core in complex with domoic acid and 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid.

More than 50 structures have been reported on the ligand-binding core of the ionotropic glutamate receptor iGluR2 that belongs to the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid-type of receptors. In contrast, the ligand-binding core of the kainic acid-type receptor iGluR5 has only been crystallized with three different ligands. Hence, additional structures of iGluR5 are needed to broaden the understanding of the ligand-binding properties of iGluR5, and the conformational changes leading to channel opening and closing. Here, we present two structures of the ligand-binding core of iGluR5; one as a complex with the partial agonist (2S,3S,4S)-3-carboxymethyl-4-[(1Z,3E,5R)-5-carboxy-1-methyl-hexa-1,3-dienyl]-pyrrolidine-2-carboxylic acid (domoic acid) and one as a complex with the antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid ((S)-ATPO). In agreement with the partial agonist activity of domoic acid, the ligand-binding core of the iGluR5 complex is stabilized by domoic acid in a conformation that is 11 degrees more open than the conformation observed in the full agonist (S)-glutamic acid complex. This is primarily caused by the 5-carboxy-1-methyl-hexa-1,3-dienyl moiety of domoic acid and residues Val685-Thr690 of iGluR5. An even larger domain opening of 28 degrees is introduced upon binding of the antagonist (S)-ATPO. It appears that the span of domain opening is much larger in the ligand-binding core of iGluR5 (30 degrees) compared with what has been observed in iGluR2 (19 degrees ). Similarly, much larger variation in the distances between transmembrane linker residues in the two protomers comprising the dimer is observed in iGluR5 as compared with iGluR2.

An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo.

Augmentation of nicotinic alpha7 receptor function is considered to be a potential therapeutic strategy aimed at ameliorating cognitive and mnemonic dysfunction in relation to debilitating pathological conditions, such as Alzheimer's disease and schizophrenia. In the present report, a novel positive allosteric modulator of the alpha7 nicotinic acetylcholine receptor (nAChR), 1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea (NS1738), is described. NS1738 was unable to displace or affect radioligand binding to the agonist binding site of nicotinic receptors, and it was devoid of effect when applied alone in electrophysiological paradigms. However, when applied in the presence of acetylcholine (ACh), NS1738 produced a marked increase in the current flowing through alpha7 nAChRs, as determined in both oocyte electrophysiology and patch-clamp recordings from mammalian cells. NS1738 acted by increasing the peak amplitude of ACh-evoked currents at all concentrations; thus, it increased the maximal efficacy of ACh. Oocyte experiments indicated an increase in ACh potency as well. NS1738 had only marginal effects on the desensitization kinetics of alpha7 nAChRs, as determined from patch-clamp studies of both transfected cells and cultured hippocampal neurons. NS1738 was modestly brain-penetrant, and it was demonstrated to counteract a (-)-scopolamine-induced deficit in acquisition of a water-maze learning task in rats. Moreover, NS1738 improved performance in the rat social recognition test to the same extent as (-)-nicotine, demonstrating that NS1738 is capable of producing cognitive enhancement in vivo. These data support the notion that alpha7 nAChR allosteric modulation may constitute a novel pharmacological principle for the treatment of cognitive dysfunction.

Mutational studies using a cation-conducting GABAA receptor reveal the selectivity determinants of the Cys-loop family of ligand-gated ion channels.

The present study evaluates how four key amino acid residue positions (- 4' to - 1') within the M1-M2 linker of the GABA(A) receptor beta subunit influences ion selectivity of a cation-conducting GABA receptor. Cation selectivity was found to be highly dependent on the side-chains of the amino acid residues present. The critical factor for cation selectivity was the presence of a negatively charged Glu or Asp residue in the -1' position. Receptors containing the neutral amino acids Gln or Asn or a positively charged Arg residue were anion selective. In the presence of a -1' Glu residue, the amino acids in adjacent positions were also found to be important determinants of cation selectivity. Moreover, the length of the M1-M2 linker as well as the presence of a Pro residue within this segment also affected ion selectivity, suggesting that the local environment and three-dimensional position of the -1' Glu are essential determinants of cation permeation. Conversely, no specific amino acid residues were found to be essential for anion selectivity, suggesting that the basic architecture of the selectivity segment of this class of receptor channels is optimally suited for anion conduction.

pH Dependency and desensitization kinetics of heterologously expressed combinations of acid-sensing ion channel subunits.

The exact subunit combinations of functional native acid-sensing ion channels (ASICs) have not been established yet, but both homomeric and heteromeric channels are likely to exist. To determine the ability of different subunits to assemble into heteromeric channels, a number of ASIC1a-, ASIC1b-, ASIC2a-, ASIC2b-, and ASIC3-containing homo- and heteromeric channels were studied by whole-cell patch clamp recordings with respect to pH sensitivity, desensitization kinetics, and level of sustained current normalized to peak current. Analyzing and comparing data for these three features demonstrated unique heteromeric channels in a number of co-expression experiments. Formation of heteromeric ASIC1a+2a and ASIC1b+2a channels was foremost supported by the desensitization characteristics that were independent of proton concentration, a feature none of the respective homomeric channels has. Several lines of evidence supported formation of ASIC1a+3, ASIC1b+3, and ASIC2a+3 heteromeric channels. The most compelling was the desensitization characteristics, which, besides being proton-independent, were faster than those of any of the respective homomeric channels. ASIC2b, which homomerically expressed is not activated by protons per se, did not appear to form unique heteromeric combinations with other subunits and in fact appeared to suppress the function of ASIC1b. Co-expression of three subunits such as ASIC1a+2a+3 and ASIC1b+2a+3 resulted in data that could best be explained by coexistence of multiple channel populations within the same cell. This observation seems to be in good agreement with the fact that ASIC-expressing sensory neurons display a variety of acid-evoked currents.

Distribution of high-voltage-activated calcium channels in cultured gamma-aminobutyric acidergic neurons from mouse cerebral cortex.

The localization of voltage-gated calcium channel (VGCC) alpha(1) subunits in cultured GABAergic mouse cortical neurons was examined by immunocytochemical methods. Ca(v)1.2 and Ca(v)1.3 subunits of L-type VGCCs were found in cell bodies and dendrites of GABA-immunopositive neurons. Likewise, the Ca(v)2.3 subunit of R-type VGCCs was expressed in a somatodendritic pattern. Ca(v)2.2 subunits of N-type channels were found exclusively in small varicosities that were identified as presynaptic nerve terminals based on their expression of synaptic marker proteins. Two splice variants of the Ca(v)2.1 subunit of P/Q-type VGCCs showed widely differing expression patterns. The rbA isoform displayed a purely somatodendritic staining pattern, whereas the BI isoform was confined to axon-like fibers and nerve terminals. The nerve terminals of these cultured GABAergic neurons express Ca(v)2.2 either alone or in combination with Ca(v)2.1 (BI isoform) but never express Ca(v)2.1 alone. The functional association between VGCCs and the neurotransmitter release machinery was probed using the FM1-43 dye-labeling technique. N-type VGCCs were found to be tightly coupled to exocytosis in these cultured cortical neurons, and P-type VGCCs were also important in a fraction of the cells. The predominant role of N-type VGCCs in neurotransmitter release and the specific localization of the BI isoform of Ca(v)2.1 in the nerve terminals of these neurons distinguish them from previously studied central neurons. The complementary localization patterns observed for two different isoforms of the Ca(v)2.1 subunits provide direct evidence for alternative splicing as a means of generating functional diversity among neuronal calcium channels.

The beta subunit determines the ion selectivity of the GABAA receptor.

The gamma-aminobutyric acid, type A (GABA(A)) receptor is a chloride-conducting receptor composed of alpha, beta, and gamma subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1-M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1-M2 loop of the beta(3) subunit to the corresponding amino acids of the alpha(7) nicotinic acetylcholine subunit rendered the GABA(A) receptor cation-selective upon co-expression with wild type alpha(2) and gamma(2) subunits. Similar mutations in the alpha(2) or gamma(2) subunits did not lead to such a change in ion selectivity. This suggests a unique role for the beta(3) subunit in determining the ion selectivity of the GABA(A) receptor. The pharmacology of the mutated GABA(A) receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn(2+) and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC(50) = 1.3 microm) compared with the wild type receptor (EC(50) = 6.4 microm) and a loss of desensitization to GABA of the mutant receptor.