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Trigeminal neuralgia, historically dubbed the "suicide disease," is an exceedingly painful neurologic condition characterized by sudden episodes of intense facial pain. Unfortunately, the only U.S. Food and Drug Administration (FDA)-approved medication for trigeminal neuralgia carries substantial side effects, with many patients requiring surgery. Here, we identify the NRF2 transcriptional network as a potential therapeutic target. We report that cerebrospinal fluid from patients with trigeminal neuralgia accumulates reactive oxygen species, several of which directly activate the pain-transducing channel TRPA1. Similar to our patient cohort, a mouse model of trigeminal neuropathic pain also exhibits notable oxidative stress. We discover that stimulating the NRF2 antioxidant transcriptional network is as analgesic as inhibiting TRPA1, in part by reversing the underlying oxidative stress. Using a transcriptome-guided drug discovery strategy, we identify two NRF2 network modulators as potential treatments. One of these candidates, exemestane, is already FDA-approved and may thus be a promising alternative treatment for trigeminal neuropathic pain.

Synapses connect discrete neurons into vast networks that send, receive, and encode diverse forms of information. Synaptic function and plasticity, the neuronal process of adapting to diverse and variable inputs, depend on the dynamic nature of synaptic molecular components, which is mediated in part by cell adhesion signaling pathways. Here, we found that the enzyme biliverdin reductase (BVR) physically links together key focal adhesion signaling molecules at the synapse. BVR -null ( BVR -/- ) mice exhibited substantial deficits in learning and memory on neurocognitive tests, and hippocampal slices in which BVR was postsynaptically depleted showed deficits in electrophysiological responses to stimuli. RNA sequencing, biochemistry, and pathway analyses suggested that these deficits were mediated through the loss of focal adhesion signaling at both the transcriptional and biochemical level in the hippocampus. Independently of its catalytic function, BVR acted as a bridge between the primary focal adhesion signaling kinases FAK and Pyk2 and the effector kinase Src. Without BVR, FAK and Pyk2 did not bind to and stimulate Src, which then did not phosphorylate the N -methyl-d-aspartate (NMDA) receptor, a critical posttranslational modification for synaptic plasticity. Src itself is a molecular hub on which many signaling pathways converge to stimulate NMDAR-mediated neurotransmission, thus positioning BVR at a prominent intersection of synaptic signaling.

Cocaine exerts its stimulant effect by inhibiting dopamine (DA) reuptake, leading to increased dopamine signaling. This action is thought to reflect the binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share cocaine’s behavioral actions. Further, recent reports show more potent actions of the drug, implying the existence of a high-affinity receptor for cocaine. We now report high-affinity binding of cocaine associated with the brain acid soluble protein 1 (BASP1) with a dissociation constant (Kd) of 7 nM. Knocking down BASP1 in the striatum inhibits [3H]cocaine binding to striatal synaptosomes. Depleting BASP1 in the nucleus accumbens but not the dorsal striatum diminishes locomotor stimulation in mice. Our findings imply that BASP1 is a pharmacologically relevant receptor for cocaine.

Alzheimer's disease (AD), the most common cause of dementia and neurodegeneration in the elderly, is characterized by deterioration of memory and executive and motor functions. Neuropathologic hallmarks of AD include neurofibrillary tangles (NFTs), paired helical filaments, and amyloid plaques. Mutations in the microtubule-associated protein Tau, a major component of the NFTs, cause its hyperphosphorylation in AD. We have shown that signaling by the gaseous molecule hydrogen sulfide (H 2 S) is dysregulated during aging. H 2 S signals via a posttranslational modification termed sulfhydration/persulfidation, which participates in diverse cellular processes. Here we show that cystathionine γ-lyase (CSE), the biosynthetic enzyme for H 2 S, binds wild type Tau, which enhances its catalytic activity. By contrast, CSE fails to bind Tau P301L, a mutant that is present in the 3xTg-AD mouse model of AD. We further show that CSE is depleted in 3xTg-AD mice as well as in human AD brains, and that H 2 S prevents hyperphosphorylation of Tau by sulfhydrating its kinase, glycogen synthase kinase 3β (GSK3β). Finally, we demonstrate that sulfhydration is diminished in AD, while administering the H 2 S donor sodium GYY4137 (NaGYY) to 3xTg-AD mice ameliorates motor and cognitive deficits in AD., Competing Interests: Competing interest statement: M.W., R.T., and the University of Exeter have patents (awarded/pending) on hydrogen sulfide delivery molecules and their therapeutic use.

Bilirubin is one of the most frequently measured metabolites in medicine, yet its physiologic roles remain unclear. Bilirubin can act as an antioxidant in vitro, but whether its redox activity is physiologically relevant is unclear because many other antioxidants are far more abundant in vivo. Here, we report that depleting endogenous bilirubin renders mice hypersensitive to oxidative stress. We find that mice lacking bilirubin are particularly vulnerable to superoxide (O 2 ⋅- ) over other tested reactive oxidants and electrophiles. Whereas major antioxidants such as glutathione and cysteine exhibit little to no reactivity toward O 2 ⋅- , bilirubin readily scavenges O 2 ⋅- . We find that bilirubin's redox activity is particularly important in the brain, where it prevents excitotoxicity and neuronal death by scavenging O 2 ⋅- during NMDA neurotransmission. Bilirubin's unique redox activity toward O 2 ⋅- may underlie a prominent physiologic role despite being significantly less abundant than other endogenous and exogenous antioxidants., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes ranging from learning and memory to stroke. The tripeptide, glutathione, is one-third glutamate and present at up to low millimolar intracellular concentrations in brain, mediating antioxidant defenses and drug detoxification. Because of the substantial amounts of brain glutathione and its rapid turnover under homeostatic control, we hypothesized that glutathione is a relevant reservoir of glutamate and could influence synaptic excitability. We find that drugs that inhibit generation of glutamate by the glutathione cycle elicit decreases in cytosolic glutamate and decreased miniature excitatory postsynaptic potential (mEPSC) frequency. In contrast, pharmacologically decreasing the biosynthesis of glutathione leads to increases in cytosolic glutamate and enhanced mEPSC frequency. The glutathione cycle can compensate for decreased excitatory neurotransmission when the glutamate-glutamine shuttle is inhibited. Glutathione may be a physiologic reservoir of glutamate neurotransmitter., Competing Interests: The authors declare no conflict of interest.

Activated immune cells undergo a metabolic switch to aerobic glycolysis akin to the Warburg effect, thereby presenting a potential therapeutic target in autoimmune disease. Dimethyl fumarate (DMF), a derivative of the Krebs cycle intermediate fumarate, is an immunomodulatory drug used to treat multiple sclerosis and psoriasis. Although its therapeutic mechanism remains uncertain, DMF covalently modifies cysteine residues in a process termed succination. We found that DMF succinates and inactivates the catalytic cysteine of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in mice and humans, both in vitro and in vivo. It thereby down-regulates aerobic glycolysis in activated myeloid and lymphoid cells, which mediates its anti-inflammatory effects. Our results provide mechanistic insight into immune modulation by DMF and represent a proof of concept that aerobic glycolysis is a therapeutic target in autoimmunity., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

ATM drives DNA repair by phosphorylating the histone variant H2AX. While ATM mutations elicit prominent neurobehavioral phenotypes, neural roles for H2AX have been elusive. We report impaired motor learning and balance in H2AX-deficient mice. Mitigation of reactive oxygen species (ROS) with N-acetylcysteine (NAC) reverses the behavioral deficits. Mouse embryonic fibroblasts deficient for H2AX exhibit increased ROS production and failure to activate the antioxidant response pathway controlled by the transcription factor NRF2. The NRF2 targets GCLC and NQO1 are depleted in the striatum of H2AX knockouts, one of the regions most vulnerable to ROS-mediated damage. These findings establish a role for ROS in the behavioral deficits of H2AX knockout mice and reveal a physiologic function of H2AX in mediating influences of oxidative stress on NRF2-transcriptional targets and behavior.

Rationale: Inositol polyphosphate multikinase (IPMK) and its major product inositol pentakisphosphate (IP5) regulate a variety of cellular functions, but their role in vascular biology remains unexplored., Objective: We have investigated the role of IPMK in regulating angiogenesis., Methods and Results: Deletion of IPMK in fibroblasts induces angiogenesis in both in vitro and in vivo models. IPMK deletion elicits a substantial increase of VEGF (vascular endothelial growth factor), which mediates the regulation of angiogenesis by IPMK. The regulation of VEGF by IPMK requires its catalytic activity. IPMK is predominantly nuclear and regulates gene transcription. However, IPMK does not apparently serve as a transcription factor for VEGF. HIF (hypoxia-inducible factor)-1α is a major determinant of angiogenesis and induces VEGF transcription. IPMK deletion elicits a major enrichment of HIF-1α protein and thus VEGF. HIF-1α is constitutively ubiquitinated by pVHL (von Hippel-Lindau protein) followed by proteasomal degradation under normal conditions. However, HIF-1α is not recognized and ubiquitinated by pVHL in IPMK KO (knockout) cells. IP5 reinstates the interaction of HIF-1α and pVHL. HIF-1α prolyl hydroxylation, which is prerequisite for pVHL recognition, is interrupted in IPMK-deleted cells. IP5 promotes HIF-1α prolyl hydroxylation and thus pVHL-dependent degradation of HIF-1α. Deletion of IPMK in mouse brain increases HIF-1α/VEGF levels and vascularization. The increased VEGF in IPMK KO disrupts blood-brain barrier and enhances brain blood vessel permeability., Conclusions: IPMK, via its product IP5, negatively regulates angiogenesis by inhibiting VEGF expression. IP5 acts by enhancing HIF-1α hydroxylation and thus pVHL-dependent degradation of HIF-1α., (© 2017 American Heart Association, Inc.)

Inositol hexakisphosphate kinase 1 (IP6K1), which generates 5-diphosphoinositol pentakisphosphate (5-IP7), physiologically mediates numerous functions. We report that IP6K1 deletion leads to brain malformation and abnormalities of neuronal migration. IP6K1 physiologically associates with α-actinin and localizes to focal adhesions. IP6K1 deletion disrupts α-actinin's intracellular localization and function. The IP6K1 deleted cells display substantial decreases of stress fiber formation and impaired cell migration and spreading. Regulation of α-actinin by IP6K1 requires its kinase activity. Deletion of IP6K1 abolishes α-actinin tyrosine phosphorylation, which is known to be regulated by focal adhesion kinase (FAK). FAK phosphorylation is substantially decreased in IP6K1 deleted cells. 5-IP7, a product of IP6K1, promotes FAK autophosphorylation. Pharmacologic inhibition of IP6K by TNP [N2-( m -Trifluorobenzyl), N6-( p -nitrobenzyl)purine] recapitulates the phenotype of IP6K1 deletion. These findings establish that IP6K1 physiologically regulates neuronal migration by binding to α-actinin and influencing phosphorylation of both FAK and α-actinin through its product 5-IP7.

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells., Significance Statement: We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function., (Copyright © 2015 the authors 0270-6474/15/3511056-12$15.00/0.)

Huntington's disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington's disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington's disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington's disease tissues, which may mediate Huntington's disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington's disease tissues and in intact mouse models of Huntington's disease, suggesting therapeutic potential.

The tumor suppressor protein p53 is a critical stress response transcription factor that induces the expression of genes leading to cell cycle arrest, apoptosis, and tumor suppression. We found that mammalian inositol polyphosphate multikinase (IPMK) stimulated p53-mediated transcription by binding to p53 and enhancing its acetylation by the acetyltransferase p300 independently of its inositol phosphate and lipid kinase activities. Genetic or RNA interference (RNAi)-mediated knockdown of IPMK resulted in decreased activation of p53, decreased recruitment of p53 and p300 to target gene promoters, abrogated transcription of p53 target genes, and enhanced cell viability. Additionally, blocking the IPMK-p53 interaction decreased the extent of p53-mediated transcription. These results suggest that IPMK acts as a transcriptional coactivator for p53 and that it is an integral part of the p53 transcriptional complex facilitating cell death.

Increases in S-nitrosylation and inactivation of the neuroprotective ubiquitin E3 ligase, parkin, in the brains of patients with Parkinson's disease are thought to be pathogenic and suggest a possible mechanism linking parkin to sporadic Parkinson's disease. Here we demonstrate that physiologic modification of parkin by hydrogen sulfide, termed sulfhydration, enhances its catalytic activity. Sulfhydration sites are identified by mass spectrometry analysis and are investigated by site-directed mutagenesis. Parkin sulfhydration is markedly depleted in the brains of patients with Parkinson's disease, suggesting that this loss may be pathologic. This implies that hydrogen sulfide donors may be therapeutic.

The inositol pyrophosphate, diphosphoinositol pentakisphosphate, regulates p53 and protein kinase Akt signaling, and its aberrant increase in cells has been implicated in apoptosis and insulin resistance. Inositol hexakisphosphate kinase-2 (IP6K2), one of the major inositol pyrophosphate synthesizing enzymes, mediates p53-linked apoptotic cell death. Casein kinase-2 (CK2) promotes cell survival and is upregulated in tumors. We show that CK2 mediated cell survival involves IP6K2 destabilization. CK2 physiologically phosphorylates IP6K2 at amino acid residues S347 and S356 contained within a PEST sequence, a consensus site for ubiquitination. HCT116 cells depleted of IP6K2 are resistant to cell death elicited by CK2 inhibitors. CK2 phosphorylation at the degradation motif of IP6K2 enhances its ubiquitination and subsequent degradation. IP6K2 mutants at the CK2 sites that are resistant to CK2 phosphorylation are metabolically stable.

mTOR complex 1 (mTORC1; mammalian target of rapamycin [mTOR] in complex with raptor) is a key regulator of protein synthesis and cell growth in response to nutrient amino acids. Here we report that inositol polyphosphate multikinase (IPMK), which possesses both inositol phosphate kinase and lipid kinase activities, regulates amino acid signaling to mTORC1. This regulation is independent of IPMK's catalytic function, instead reflecting its binding with mTOR and raptor, which maintains the mTOR-raptor association. Thus, IPMK appears to be a physiologic mTOR cofactor, serving as a determinant of mTORC1 stability and amino acid-induced mTOR signaling. Substances that block IPMK-mTORC1 binding may afford therapeutic benefit in nutrient amino acid-regulated conditions such as obesity and diabetes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase that regulates glucose homeostasis and protein translation, respectively, via the GSK3β and mTOR pathways. Thus, Akt and mTOR signaling are dramatically augmented and GSK3β signaling reduced in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 affects this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby affecting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high-fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Inositol pyrophosphates have been implicated in numerous biological processes. Inositol hexakisphosphate kinase-2 (IP6K2), which generates the inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP7), influences apoptotic cell death. The tumor suppressor p53 responds to genotoxic stress by engaging a transcriptional program leading to cell-cycle arrest or apoptosis. We demonstrate that IP6K2 is required for p53-mediated apoptosis and modulates the outcome of the p53 response. Gene disruption of IP6K2 in colorectal cancer cells selectively impairs p53-mediated apoptosis, instead favoring cell-cycle arrest. IP6K2 acts by binding directly to p53 and decreasing expression of proarrest gene targets such as the cyclin-dependent kinase inhibitor p21.

S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.

Heat-shock proteins (HSPs) are abundant, inducible proteins best known for their ability to maintain the conformation of proteins and to refold damaged proteins. Some HSPs, especially HSP90, can be antiapoptotic and the targets of anticancer drugs. Inositol hexakisphosphate kinase-2 (IP6K2), one of a family of enzymes generating the inositol pyrophosphate IP7 [diphosphoinositol pentakisphosphate (5-PP-IP5)], mediates apoptosis. Increased IP6K2 activity sensitizes cancer cells to stressors, whereas its depletion blocks cell death. We now show that HSP90 physiologically binds IP6K2 and inhibits its catalytic activity. Drugs and selective mutations that abolish HSP90-IP6K2 binding elicit activation of IP6K2, leading to cell death. Thus, the prosurvival actions of HSP90 reflect the inhibition of IP6K2, suggesting that selectively blocking this interaction could provide effective and safer modes of chemotherapy.

In a previous study, we showed that the inositol pyrophosphate diphosphoinositol pentakisphosphate (IP(7)) physiologically phosphorylates mammalian and yeast proteins. We now report that this phosphate transfer reflects pyrophosphorylation. Thus, proteins must be prephosphorylated by ATP to prime them for IP(7) phosphorylation. IP(7) phosphorylates synthetic phosphopeptides but not if their phosphates have been masked by methylation or pyrophosphorylation. Moreover, IP(7) phosphorylated peptides are more acid-labile and more resistant to phosphatases than ATP phosphorylated peptides, indicating a different type of phosphate bond. Pyrophosphorylation may represent a novel mode of signaling to proteins.

The atypical antipsychotic drugs (AAPDs) have markedly enhanced the treatment of schizophrenias but their use has been hindered by the major weight gain elicited by some AAPDs. We report that orexigenic AAPDs potently and selectively activate hypothalamic AMP-kinase, an action abolished in mice with deletion of histamine H1 receptors. These findings may afford a means of developing more effective therapeutic agents and provide insight into the hypothalamic regulation of food intake.

Phosphatidylinositol 3,4,5-trisphosphate is a major intracellular messenger molecule thought to be formed almost exclusively by cytosolic, wortmannin-inhibited phosphoinositide 3-kinase family members. Inositol polyphosphate multikinase was identified as an enzyme that generates a series of water-soluble inositol phosphates. We now report the robust, physiologic, and evolutionarily conserved phosphoinositide 3-kinase activity of inositol polyphosphate multikinase, which is localized to nuclei and unaffected by wortmannin. In yeast, this inositol lipid kinase activity physiologically regulates transcription.

Inositol pyrophosphates physiologically regulate vesicular endocytosis, ribosomal disposition, and directly phosphorylate proteins. Here we demonstrate roles in cell death and regulation of telomere length. Lethal actions of wortmannin and caffeine are selectively abolished in yeast mutants that cannot synthesize inositol pyrophosphates. Wortmannin and caffeine appear to act through the phosphoinositide 3-kinase-related protein kinases Tel1 and Mec1, known regulators of telomere length. Inositol pyrophosphates physiologically antagonize the actions of these kinases, which is demonstrated by the fact that yeast mutants with reduced or elevated levels of inositol pyrophosphates, respectively, display longer and shorter telomeres.

The inositol pyrophosphates IP7 and IP8 contain highly energetic pyrophosphate bonds. Although implicated in various biologic functions, their molecular sites of action have not been clarified. Using radiolabeled IP7, we detected phosphorylation of multiple eukaryotic proteins. We also observed phosphorylation of endogenous proteins by endogenous IP7 in yeast. Phosphorylation by IP7 is nonenzymatic and may represent a novel intracellular signaling mechanism.

The specificity of the reactions of nitric oxide (NO) with its neuronal targets is determined in part by the precise localizations of neuronal NO synthase (nNOS) within the cell. The targeting of nNOS is mediated by adapter proteins that interact with its PDZ domain. Here, we show that the nNOS adapter protein, CAPON, interacts with synapsins I, II, and III through an N-terminal phosphotyrosine-binding domain interaction, which leads to a ternary complex comprising nNOS, CAPON, and synapsin I. The significance of this ternary complex is demonstrated by changes in subcellular localization of nNOS in mice harboring genomic deletions of both synapsin I and synapsin II. These results suggest a mechanism for specific actions of NO at presynaptic sites.

The inositol pyrophosphate disphosphoinositol pentakisphosphate (PP-InsP(3)/InsP(7)) is formed in mammals by two recently cloned inositol hexakiphosphate kinases, InsP(6)K1 and InsP(6)K2 (Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326). We now report the identification, cloning, and characterization of a third InsP(7) forming enzyme designated InsP(6)K3. InsP(6)K3 displays 50 and 45% sequence identity to InsP(6)K1 and InsP(6)K2, respectively, with a smaller mass (46 kDa) and a more basic character than the other two enzymes. InsP(6)K3 is most enriched in the brain where its localization resembles InsP(6)K1 and InsP(6)K2. Intracellular disposition discriminates the three enzymes with InsP(6)K2 being exclusively nuclear, InsP(6)K3 predominating in the cytoplasm, and InsP(6)K1 displaying comparable nuclear and cytosolic densities.

Using a consensus sequence in inositol phosphate kinase, we have identified and cloned a 44-kDa mammalian inositol phosphate kinase with broader catalytic capacities than any other member of the family and which we designate mammalian inositol phosphate multikinase (mIPMK). By phosphorylating inositol 4,5-bisphosphate, mIPMK provides an alternative biosynthesis for inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]. mIPMK also can form the pyrophosphate disphosphoinositol tetrakisphosphate (PP-InsP(4)) from InsP(5). Additionally, mIPMK forms InsP(4) from Ins(1,4,5)P(3) and InsP(5) from Ins(1,3,4,5)P(4).

Inositol (1,4,5) trisphosphate (Ins(1,4,5)P(3)) is a well-known messenger molecule that releases calcium from intracellular stores. Homologues with up to six phosphates have been characterized and recently, homologues with seven or eight phosphate groups, including pyrophosphates, have been identified. These homologues are diphosphoinositol pentakisphosphate (PP-InsP(5)/InsP(7)) and bis(diphospho)inositol tetrakisphosphate (bis-PP-InsP(4)/InsP(8)) [1], the rapid turnover of which [2] is regulated by calcium [2] and adrenergic receptor activity [3]. It has been proposed that the high-energy pyrophosphates might participate in protein phosphorylation [4]. We have purified InsP(6) kinase [5] and PP-InsP(5) kinase [6], both of which display ATP synthase activity, transferring phosphate to ADP. Here, we report the cloning of two mammalian InsP(6) kinases and a yeast InsP(6) kinase. Furthermore, we show that the yeast protein, ArgRIII, is an inositol-polyphosphate kinase that can convert InsP(3) to InsP(4), InsP(5) and InsP(6). We have identified a new family of highly conserved inositol-polyphosphate kinases that contain a newly identified, unique consensus sequence.

Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is important for N-methyl-D-aspartate (NMDA) receptor-dependent neurotransmitter release, neurotoxicity, and cyclic GMP elevations. The coupling of NMDA receptor-mediated calcium influx and nNOS activation is postulated to be due to a physical coupling of the receptor and the enzyme by an intermediary adaptor protein, PSD95, through a unique PDZ-PDZ domain interaction between PSD95 and nNOS. Here, we report the identification of a novel nNOS-associated protein, CAPON, which is highly enriched in brain and has numerous colocalizations with nNOS. CAPON interacts with the nNOS PDZ domain through its C terminus. CAPON competes with PSD95 for interaction with nNOS, and overexpression of CAPON results in a loss of PSD95/nNOS complexes in transfected cells. CAPON may influence nNOS by regulating its ability to associate with PSD95/NMDA receptor complexes.

We have stably expressed cDNA for the rat brain Ca2+ sensing receptor in Chinese hamster ovary cells. Stimulation of phosphatidylinositol hydrolysis and arachidonic acid (AA) release displayed markedly cooperative responses to Ca2+ with Hill coefficients of 4-5. Both phosphatidylinositol and AA responses were not detected below a threshold of 1.5 mM Ca2+. Mg2+ behaved as a partial agonist with only half the maximal inositol phosphate and AA responses displayed by Ca2+ and with a more shallow concentration-response slope. The potency of Mg2+ in augmenting inositol phosphate and AA responses, in the presence of 1.5 mM Ca2+, implies that serum Mg2+ concentrations attained in clinical conditions will influence the Ca2+-sensing receptor.

Background: Clozapine, the classic atypical neuroleptic, exerts therapeutic actions in schizophrenic patients unresponsive to most neuroleptics. Clozapine interacts with numerous neurotransmitter receptors, and selective actions at novel subtypes of dopamine and serotonin receptors have been proposed to explain clozapine's unique psychotropic effects. To identify sites with which clozapine preferentially interacts in a therapeutic setting, we have characterized clozapine binding to brain membranes., Materials and Methods: [3H]Clozapine binding was examined in rat brain membranes as well as cloned-expressed 5-HT6 serotonin receptors., Results: [3H]Clozapine binds with low nanomolar affinity to two distinct sites. One reflects muscarinic receptors consistent with the drug's anticholinergic actions. The drug competition profile of the second site most closely resembles 5HT6 serotonin receptors, though serotonin itself displays low affinity. [3H]Clozapine binding levels are similar in all brain regions examined with no concentration in the corpus striatum., Conclusions: Besides muscarinic receptors, clozapine primarily labels sites with properties resembling 5HT6 serotonin receptors. If this is also the site with which clozapine principally interacts in intact human brain, it may account for the unique beneficial actions of clozapine and other atypical neuroleptics, and provide a molecular target for developing new, safer, and more effective agents.

We have molecularly cloned a calcium sensing receptor (CaSR) from a rat striatal cDNA library. Rat CaSR displays 92% overall homology to its bovine counterpart with seven putative transmembrane domains characteristic of the superfamily of guanine nucleotide-binding proteins and significant homology with the metabotropic glutamate receptors. Northern blot analysis reveals two transcripts in thyroid, kidney, lung, ileum, and pituitary. In brain highest regional expression of the RNA occurs in the hypothalamus and the corpus striatum. Immunohistochemistry reveals discrete punctate localizations throughout the brain that appear to be associated with nerve terminals. No staining is evident in cell bodies of neurons or glia. Cerebral arteries display an intense network of CaSR immunoreactive fibers associated with vessel innervation. CaSR on nerve terminal membranes may regulate neurotransmitter disposition in response to Ca2+ levels in the synaptic space.

Modulation of the neuronal omega-conotoxin GVIA-sensitive N-type voltage-dependent calcium channel (VDCC) by neurotransmitters and guanine nucleotides suggests a dynamic interaction between activated G-protein alpha subunits and the N-type VDCC. Our previous report on the purification of the N-type VDCC (McEnery, M. W., Snowman, A. M., Sharp, A. H., Adams, M. E., and Snyder, S. H. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11095-11099), has led us to investigate a possible association of CTXR with an endogenous G alpha subunit. The addition of the G-protein activator AIF4- modulated the 125I-CTX binding characteristics of the solubilized CTXR. Further immunological analyses employing G alpha subunit-specific antibodies to monitor the cofractionation of G alpha with 125I-CTX binding activity throughout the purification procedure indicate the selective recovery of Go alpha in the purified CTXR preparation, as neither Gs alpha, Gi alpha, nor G beta gamma could be detected. Furthermore, Go alpha associated with CTXR acted as a substrate for pertussis toxin-dependent ADP-ribosylation only upon the addition of exogenous G beta gamma subunits. These results strongly indicate a high affinity complex between an activated Go alpha and CTXR maintained throughout biochemical purification of the 125I-CTX receptor.

The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC 1.14.13.39) has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct, expresses little luciferase activity when stimulated by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or both. Maximal expression depends on two discrete regulatory regions upstream of the putative TATA box. Region I (position -48 to -209) increases luciferase activity approximately 75-fold over the minimal promoter construct. Region I contains LPS-related responsive elements, including a binding site for nuclear factor interleukin 6 (NF-IL6) and the kappa B binding site for NF-kappa B, suggesting that this region regulates LPS-induced expression of the mac-NOS gene. Region II (position -913 to -1029) alone does not increase luciferase expression, but together with region I it causes an additional 10-fold increase in expression. Together the two regions increase expression 750-fold over activity obtained from a minimal promoter construct. Region II contains motifs for binding IFN-related transcription factors and thus probably is responsible for IFN-mediated regulation of LPS-induced mac-NOS. Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, how IFN-gamma augments the inflammatory response to LPS.

The immunophilins cyclophilin and FK506 binding protein (FKBP) are small, predominantly soluble proteins that bind the immunosuppressant drugs cyclosporin A and FK506, respectively, with high affinity, and which seem to mediate their pharmacological actions. The Ca(2+)-dependent protein phosphatase, calcineurin, binds the cyclophilin-cyclosporin A and FKBP-FK506 complexes, indicating that calcineurin might mediate the actions of these drugs. A physiological role for the immunophilins in the nervous system is implied by a close homology between the structure of NINA A, a protein in the neural retina of Drosophila, and cyclophilin, as well as by the high density of FKBP messenger RNA in brain tissue. Here we report that the levels of FKBP and mRNA in rat brain are extraordinarily high and that their regional localization is virtually identical to that of calcineurin, indicating that there may be a physiological link between calcineurin and the immunophilins. We also show that at low concentrations FK506 and cyclosporin A enhance the phosphorylation of endogenous protein substrates in brain tissue and in intact PC12 cells, indicating that these drugs may inhibit phosphatase activity by interacting with the immunophilin-calcineurin complexes.

The mitochondrial benzodiazepine receptor (mBzR) has been solubilized with retention of reversible ligand binding, and the associated subunits were characterized. mBzR comprises immunologically distinct protein subunits of 18-, 30-, and 32-kDa. The 18-kDa protein is labeled by the isoquinoline carboxamide mBzR ligand [3H]PK14105, whereas the 30- and 32-kDa subunits are labeled by the benzodiazepine (Bz) ligands [3H]flunitrazepam and [3H]AHN-086. Selective antibodies and reagents identify the 32- and 30-kDa proteins as the voltage-dependent anion channel (VDAC) and the adenine nucleotide carrier (ADC), respectively. While isoquinoline carboxamide and Bz ligands target different subunits, they interact allosterically, as the binding of Bz and isoquinoline carboxamide ligands is mutually competitive at low nanomolar concentrations. Moreover, eosin-5-maleimide and mercuric chloride inhibit [3H]PK11195 binding to the intact receptor via sulfhydryl groups that are present in ADC. VDAC and ADC, outer and inner mitochondrial membrane channel proteins, respectively, together with the 18-kDa subunit, may comprise mBzR at functionally important transport sites at the junction of two mitochondrial membranes.

The omega-conotoxin GVIA (CTX) receptor has been purified 1900-fold to apparent homogeneity by monitoring both reversible binding of 125I-labeled CTX (125I-CTX) and photoincorporation of N-hydroxysuccinimidyl-4-azidobenzoate-125I-CTX (HSA-125I-CTX). Photoincorporation of HSA-125I-CTX into a 230-kDa protein exhibits a pharmacologic and chromatographic profile indicating that the 230-kDa protein is the CTX-binding subunit of the receptor. The pharmacologic specificity of 125I-CTX binding to the purified CTX receptor closely resembles that of the native membrane-bound form with respect to sensitivity towards CTX (Kd = 32 pM) and other peptide toxin antagonists. The purified CTX receptor comprises the 230-kDa protein (alpha 1) and four additional proteins with apparent molecular masses of 140 (alpha 2), 110, 70 (beta 2), and 60 (beta 1) kDa. This subunit structure closely resembles that of the 1,4-dihydropyridine-sensitive L-type calcium channel.

Opipramol (OP), a clinically effective antidepressant with a tricyclic structure, is inactive as an inhibitor of biogenic amine uptake. [3H]Opipramol binds saturably to rat brain membranes (apparent KD = 4 nM, Bmax = 3 pmol/mg of protein). [3H]Opipramol binding can be differentiated into haloperidol-sensitive and -resistant components, with Ki values for haloperidol of 1 nM (Bmax = 1 pmol/mg of protein) and 350 nM (Bmax = 1.9 pmol/mg of protein), respectively. The drug specificity of the haloperidol-sensitive component is the same as that of sigma receptors labeled with (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperdine. The haloperidol-resistant component does not correspond to any known neurotransmitter receptor or uptake recognition site. It displays high affinity for phenothiazines and related structures such as perphenazine, clopenthixol, and flupenthixol, whose potencies are comparable to that of opipramol. Because certain of these drugs are more potent at the haloperidol-resistant opipramol site than in exerting any other action, it is possible that this opipramol-selective site may mediate their therapeutic effects.

First-generation H1-antagonist antihistamines, such as hydroxyzine, have the ability to cross the blood-brain barrier and cause sedation, which limits their usefulness in the treatment of allergic disorders. Cetirizine, a carboxylated metabolite of hydroxyzine, possesses the parent compound's antihistaminic activity but causes less sedation. We compared the activity of cetirizine at central H1 sites with that of hydroxyzine and terfenadine. We also compared the ability of cetirizine and three antihistamines to cross the blood-brain barrier. In each case we found that the drug's potency at H1 receptors in the central nervous system was similar to its activity in displacing H1 receptors in the lung. However, the selectivity for H1 receptors varied widely from drug to drug. Cetirizine did not bind at any of the receptors investigated, except H1 sites, even at concentrations as high as 10 mumol/L. Hydroxyzine and dexchlorpheniramine and, to a lesser extent, terfenadine crossed the blood-brain barrier in significant amounts. Cetirizine passed into the central nervous system only half as readily as terfenadine. These findings suggest that cetirizine's low incidence of sedative effects is most likely caused by its diminished potential to cross the blood-brain barrier and also may be partly the result of its greater selectivity for H1 receptors, compared with its effect at other receptors that may be involved in sedation.

We have characterized the odorant binding properties of purified bovine odorant-binding protein (OBP) using as a ligand [3H]3,7-dimethyloctan-1-ol ([3H]DMO). A broad variety of odorants, including terpenes, aldehydes, esters, and musks, bind to OBP with affinities of 0.2 to 100 microM. Odorant affinities for OBP correlate most closely with their stimulation of an odorant-sensitive adenylyl cyclase as well as hydrophobicity. We also measured the kinetics of binding for the ligands, [3H]DMO and 2-isobutyl-3-[3H]methoxypyrazine. Dissociation of both is markedly accelerated in the presence of excess unlabeled ligand. Competition curves of displacers for [3H]DMO binding are shallow, and saturation binding isotherms for 3H-odorants are curvilinear. These kinetic and equilibrium binding properties suggest that OBP interactions with odorant ligands are negatively cooperative.

Amiodarone and its pharmacologically active metabolite desethylamiodarone have a sodium channel blocking action that explains some of their antiarrhythmic efficacy. However, the well-documented depression of the calcium channel-dependent sinus node and atrioventricular node function that occurs with amiodarone therapy suggests that amiodarone also blocks calcium influx through voltage-dependent calcium channels. Recent electrophysiologic data support the notion that amiodarone, but not desethylamiodarone, acts as a calcium channel antagonist. In this study, the effects of amiodarone and desethylamiodarone on calcium antagonist receptors associated with the voltage-dependent calcium channels were characterized. Amiodarone, but not its active metabolite desethylamiodarone, was a potent competitor at dihydropyridine and phenylalkylamine (verapamil-like) calcium antagonist binding sites in rat heart, brain, and skeletal and smooth muscles. Substantial inhibition of calcium antagonist binding was retained even after extensive washing of membranes and 2 days after in vivo treatment of rats with amiodarone. The pattern of inhibition of calcium antagonist binding suggests that amiodarone acts at phenylalkylamine binding sites. It is suggested that the acute effects of amiodarone--sinus and atrioventricular node inhibition, vasodilatation, and negative inotropic actions--may reflect calcium antagonist influences of amiodarone itself. Chronic effects of drug therapy, such as inhibition of ventricular conduction by sodium channel blockade, may selectively involve desethylamiodarone.

Barbiturates and the related depressant drugs, etazolate and etomidate, inhibited both the binding of [3H]bicuculline methochloride (BMC) to gamma-aminobutyric acid (GABA) receptor sites and the binding of [3H] beta-carboline-3-carboxylic acid methyl ester (beta CCM) to benzodiazepine receptor sites in mammalian brain. These concentration-dependent effects were chemically specific and stereospecific in a manner correlating with the activity of barbiturates to enhance GABA responses in neurons and to enhance GABA and benzodiazepine receptor agonist binding in vitro. The barbiturate inhibition of [3H]BMC binding involved a decrease in affinity which at high concentrations of barbiturates results in an effective complete loss of detectable binding. The maximal inhibition of [3H] beta CCM binding involved a more modest decrease in affinity (increase in KD from 1.35 to 1.85 nM). The barbiturate inhibitions of both ligands could be reversed by picrotoxin, suggesting an indirect action at previously defined picrotoxin/barbiturate modulatory sites on the GABA-benzodiazepine receptor/chloride ion channel complex.

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.

The potencies of various opiates in displacing several 3H-opiate ligands' binding to rat membranes vary depending on the nature of the ligand. Whereas opiate antagonists, as well as the opioid peptides and some agonists (etorphine, levorphanol and phenazocine) display similar affinities in displacing either 3H-opiate or 3H-methionine enkephalin binding, other agonists (such as morphine and oxymorphone) are considerably (20-50 times) weaker in displacing 3H-enkephalin than 3H-dihydromorphine binding. These agonists also compete for 3H-enkephalin binding with shallow displacement curves, and are greatly weakened in displacing 3H-naloxone binding in the presence of sodium. These agonists differ from the other opiate classes by possessing a relatively hydrophilic component in their C-ring moieties which may provide a basis for the differential interactions of drugs with the opiate receptor.