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1.
Front Behav Neurosci ; 10: 136, 2016.
Article in English | MEDLINE | ID: mdl-27445731

ABSTRACT

Fragile X is the most common monogenic disorder associated with intellectual disability (ID) and autism spectrum disorders (ASD). Additionally, many patients are afflicted with executive dysfunction, ADHD, seizure disorder and sleep disturbances. Fragile X is caused by loss of FMRP expression, which is encoded by the FMR1 gene. Both the fly and mouse models of fragile X are also based on having no functional protein expression of their respective FMR1 homologs. The fly model displays well defined cognitive impairments and structural brain defects and the mouse model, although having subtle behavioral defects, has robust electrophysiological phenotypes and provides a tool to do extensive biochemical analysis of select brain regions. Decreased cAMP signaling has been observed in samples from the fly and mouse models of fragile X as well as in samples derived from human patients. Indeed, we have previously demonstrated that strategies that increase cAMP signaling can rescue short term memory in the fly model and restore DHPG induced mGluR mediated long term depression (LTD) in the hippocampus to proper levels in the mouse model (McBride et al., 2005; Choi et al., 2011, 2015). Here, we demonstrate that the same three strategies used previously with the potential to be used clinically, lithium treatment, PDE-4 inhibitor treatment or mGluR antagonist treatment can rescue long term memory in the fly model and alter the cAMP signaling pathway in the hippocampus of the mouse model.

2.
Cell Rep ; 11(5): 727-36, 2015 May 05.
Article in English | MEDLINE | ID: mdl-25921541

ABSTRACT

The PI3K enhancer PIKE links PI3K catalytic subunits to group 1 metabotropic glutamate receptors (mGlu1/5) and activates PI3K signaling. The roles of PIKE in synaptic plasticity and the etiology of mental disorders are unknown. Here, we show that increased PIKE expression is a key mediator of impaired mGlu1/5-dependent neuronal plasticity in mouse and fly models of the inherited intellectual disability fragile X syndrome (FXS). Normalizing elevated PIKE protein levels in FXS mice reversed deficits in molecular and cellular plasticity and improved behavior. Notably, PIKE reduction rescued PI3K-dependent and -independent neuronal defects in FXS. We further show that PI3K signaling is increased in a fly model of FXS and that genetic reduction of the Drosophila ortholog of PIKE, CenG1A rescued excessive PI3K signaling, mushroom body defects, and impaired short-term memory in these flies. Our results demonstrate a crucial role of increased PIKE expression in exaggerated mGlu1/5 signaling causing neuronal defects in FXS.


Subject(s)
Behavior, Animal/physiology , Fragile X Syndrome/pathology , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Gene Expression Regulation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuronal Plasticity , Animals , Brain/metabolism , Dendritic Spines/metabolism , Disease Models, Animal , Drosophila/metabolism , Drosophila Proteins/metabolism , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/metabolism , GTPase-Activating Proteins/metabolism , Mice , Mice, Knockout , Mushroom Bodies/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Biosynthesis , Receptor, Metabotropic Glutamate 5/metabolism , Receptors, Metabotropic Glutamate/metabolism , Signal Transduction
3.
J Neurosci ; 35(1): 396-408, 2015 Jan 07.
Article in English | MEDLINE | ID: mdl-25568131

ABSTRACT

Fragile X syndrome (FXS) is the leading cause of both intellectual disability and autism resulting from a single gene mutation. Previously, we characterized cognitive impairments and brain structural defects in a Drosophila model of FXS and demonstrated that these impairments were rescued by treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium. A well-documented biochemical defect observed in fly and mouse FXS models and FXS patients is low cAMP levels. cAMP levels can be regulated by mGluR signaling. Herein, we demonstrate PDE-4 inhibition as a therapeutic strategy to ameliorate memory impairments and brain structural defects in the Drosophila model of fragile X. Furthermore, we examine the effects of PDE-4 inhibition by pharmacologic treatment in the fragile X mouse model. We demonstrate that acute inhibition of PDE-4 by pharmacologic treatment in hippocampal slices rescues the enhanced mGluR-dependent LTD phenotype observed in FXS mice. Additionally, we find that chronic treatment of FXS model mice, in adulthood, also restores the level of mGluR-dependent LTD to that observed in wild-type animals. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of FXS is an important advance, in that this identifies and validates PDE-4 inhibition as potential therapeutic intervention for the treatment of individuals afflicted with FXS.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Disease Models, Animal , Fragile X Syndrome/enzymology , Neuronal Plasticity/physiology , Phosphodiesterase 4 Inhibitors/pharmacology , Animals , Animals, Genetically Modified , Cyclic Nucleotide Phosphodiesterases, Type 4/genetics , Drosophila , Female , Fragile X Syndrome/drug therapy , Fragile X Syndrome/genetics , Male , Mice , Mice, Knockout , Neuronal Plasticity/drug effects , Phosphodiesterase 4 Inhibitors/therapeutic use
4.
Drug Discov Today Technol ; 10(1): e129-36, 2013.
Article in English | MEDLINE | ID: mdl-24050241

ABSTRACT

Despite obvious differences such as the ability to fly, the fruit fly Drosophila melanogaster is similar to humans at many different levels of complexity. Studies of development, cell growth and division, metabolism and even cognition, have borne out these similarities. For example, Drosophila bearing mutations in the fly gene homologue of the known human disease fragile X are affected in fundamentally similar ways as affected humans. The ramification of this degree of similarity is that Drosophila, as a model organism, is a rich resource for learning about human cells, development and even human cognition and behavior. Drosophila has a short generation time of ten days, is cheap to propagate and maintain and has a vast array of genetic tools available to it; making Drosophila an extremely attractive organism for the study of human disease. Here, we summarize research from our lab and others using Drosophila to understand the human neurological disease, called fragile X. We focus on the Drosophila model of fragile X, its characterization, and use as a tool to identify potential drugs for the treatment of fragile X. Several clinical trials are in progress now that were motivated by this research.


Subject(s)
Disease Models, Animal , Drosophila/genetics , Fragile X Syndrome/drug therapy , Animals , Drug Evaluation, Preclinical , Fragile X Syndrome/physiopathology , Humans
5.
Front Pharmacol ; 4: 64, 2013.
Article in English | MEDLINE | ID: mdl-23720628

ABSTRACT

Metabotropic glutamate receptors (mGluRs) have well-established roles in cognition and social behavior in mammals. Whether or not these roles have been conserved throughout evolution from invertebrate species is less clear. Mammals have eight mGluRs whereas Drosophila has a single DmGluRA, which has both Gi and Gq coupled signaling activity. We have utilized Drosophila to examine the role of DmGluRA in social behavior and various phases of memory. We have found that flies that are homozygous or heterozygous for loss of function mutations of DmGluRA have impaired social behavior in male Drosophila. Futhermore, flies that are heterozygous for loss of function mutations of DmGluRA have impaired learning during training, immediate-recall memory, short-term memory, and long-term memory as young adults. This work demonstrates a role for mGluR activity in both social behavior and memory in Drosophila.

6.
Brain Res ; 1380: 106-19, 2011 Mar 22.
Article in English | MEDLINE | ID: mdl-21078304

ABSTRACT

Fragile X syndrome is the leading single gene cause of intellectual disabilities. Treatment of a Drosophila model of Fragile X syndrome with metabotropic glutamate receptor (mGluR) antagonists or lithium rescues social and cognitive impairments. A hallmark feature of the Fragile X mouse model is enhanced mGluR-dependent long-term depression (LTD) at Schaffer collateral to CA1 pyramidal synapses of the hippocampus. Here we examine the effects of chronic treatment of Fragile X mice in vivo with lithium or a group II mGluR antagonist on mGluR-LTD at CA1 synapses. We find that long-term lithium treatment initiated during development (5-6 weeks of age) and continued throughout the lifetime of the Fragile X mice until 9-11 months of age restores normal mGluR-LTD. Additionally, chronic short-term treatment beginning in adult Fragile X mice (8 weeks of age) with either lithium or an mGluR antagonist is also able to restore normal mGluR-LTD. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of Fragile X syndrome is an important advance, in that this identifies and validates these targets as potential therapeutic interventions for the treatment of individuals afflicted with Fragile X syndrome.


Subject(s)
Excitatory Amino Acid Antagonists/pharmacology , Fragile X Syndrome/drug therapy , Lithium Compounds/pharmacology , Neuronal Plasticity/drug effects , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Synaptic Transmission/drug effects , Animals , Antidepressive Agents/pharmacology , Antidepressive Agents/therapeutic use , Disease Models, Animal , Excitatory Amino Acid Antagonists/therapeutic use , Fragile X Syndrome/metabolism , Fragile X Syndrome/physiopathology , Lithium Compounds/therapeutic use , Male , Mice , Mice, Knockout , Neuronal Plasticity/genetics , Receptors, Metabotropic Glutamate/physiology , Synaptic Transmission/genetics
7.
J Neurosci ; 30(28): 9510-22, 2010 Jul 14.
Article in English | MEDLINE | ID: mdl-20631179

ABSTRACT

Alzheimer's disease (AD) is the leading cause of cognitive loss and neurodegeneration in the developed world. Although its genetic and environmental causes are not generally known, familial forms of the disease (FAD) are attributable to mutations in a single copy of the Presenilin (PS) and amyloid precursor protein genes. The dominant inheritance pattern of FAD indicates that it may be attributable to gain or change of function mutations. Studies of FAD-linked forms of presenilin (psn) in model organisms, however, indicate that they are loss of function, leading to the possibility that a reduction in PS activity might contribute to FAD and that proper psn levels are important for maintaining normal cognition throughout life. To explore this issue further, we have tested the effect of reducing psn activity during aging in Drosophila melanogaster males. We have found that flies in which the dosage of psn function is reduced by 50% display age-onset impairments in learning and memory. Treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium during the aging process prevented the onset of these deficits, and treatment of aged flies reversed the age-dependent deficits. Genetic reduction of Drosophila metabotropic glutamate receptor (DmGluRA), the inositol trisphosphate receptor (InsP(3)R), or inositol polyphosphate 1-phosphatase also prevented these age-onset cognitive deficits. These findings suggest that reduced psn activity may contribute to the age-onset cognitive loss observed with FAD. They also indicate that enhanced mGluR signaling and calcium release regulated by InsP(3)R as underlying causes of the age-dependent cognitive phenotypes observed when psn activity is reduced.


Subject(s)
Cognition/physiology , Learning/physiology , Memory/physiology , Presenilins/genetics , Age Factors , Analysis of Variance , Animals , Animals, Genetically Modified , Behavior, Animal/drug effects , Behavior, Animal/physiology , Cognition/drug effects , Courtship , Drosophila melanogaster , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Learning/drug effects , Lithium/pharmacology , Male , Memory/drug effects , Mushroom Bodies/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Presenilins/metabolism , Random Allocation , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Receptors, Metabotropic Glutamate/genetics , Receptors, Metabotropic Glutamate/metabolism
8.
J Neurosci ; 30(19): 6782-92, 2010 May 12.
Article in English | MEDLINE | ID: mdl-20463240

ABSTRACT

The diversity of protein isoforms arising from alternative splicing is thought to modulate fine-tuning of synaptic plasticity. Fragile X mental retardation protein (FMRP), a neuronal RNA binding protein, exists in isoforms as a result of alternative splicing, but the contribution of these isoforms to neural plasticity are not well understood. We show that two isoforms of Drosophila melanogaster FMRP (dFMR1) have differential roles in mediating neural development and behavior functions conferred by the dfmr1 gene. These isoforms differ in the presence of a protein interaction module that is related to prion domains and is functionally conserved between FMRPs. Expression of both isoforms is necessary for optimal performance in tests of short- and long-term memory of courtship training. The presence or absence of the protein interaction domain may govern the types of ribonucleoprotein (RNP) complexes dFMR1 assembles into, with different RNPs regulating gene expression in a manner necessary for establishing distinct phases of memory formation.


Subject(s)
Drosophila Proteins/metabolism , Fragile X Mental Retardation Protein/metabolism , Memory, Short-Term/physiology , Memory/physiology , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , Circadian Rhythm/physiology , DNA Mutational Analysis , Drosophila Proteins/genetics , Drosophila melanogaster , Female , Fragile X Mental Retardation Protein/genetics , Male , Molecular Sequence Data , Motor Activity/physiology , Neuropsychological Tests , Protein Isoforms/metabolism , Sexual Behavior, Animal/physiology , Time Factors
9.
Cell Mol Life Sci ; 67(17): 2991-3004, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20386952

ABSTRACT

Metallocarboxypeptidase D (CPD) functions in protein and peptide processing. The Drosophila CPD svr gene undergoes alternative splicing, producing forms containing 1-3 active or inactive CP domains. To investigate the function of the various CP domains, we created transgenic flies expressing specific forms of CPD in the embryonic-lethal svr (PG33) mutant. All constructs containing an active CP domain rescued the lethality with varying degrees, and full viability required inactive CP domain-3. Transgenic flies overexpressing active CP domain-1 or -2 were similar to each other and to the viable svr mutants, with pointed wing shape, enhanced ethanol sensitivity, and decreased cold sensitivity. The transgenes fully compensated for a long-term memory deficit observed in the viable svr mutants. Overexpression of CP domain-1 or -2 reduced the levels of Lys/Arg-extended adipokinetic hormone intermediates. These findings suggest that CPD domains-1 and -2 have largely redundant functions in the processing of growth factors, hormones, and neuropeptides.


Subject(s)
Drosophila Proteins/physiology , Phenotype , Protein Structure, Tertiary/physiology , Proteins/physiology , Alternative Splicing/physiology , Animals , Animals, Genetically Modified , Drosophila , Drosophila Proteins/genetics , Gene Components , Memory/physiology , Protein Structure, Tertiary/genetics , Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Wings, Animal/anatomy & histology
10.
Biogerontology ; 11(3): 347-62, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20039205

ABSTRACT

Fragile X syndrome afflicts 1 in 2,500 individuals and is the leading heritable cause of mental retardation worldwide. The overriding clinical manifestation of this disease is mild to severe cognitive impairment. Age-dependent cognitive decline has been identified in Fragile X patients, although it has not been fully characterized nor examined in animal models. A Drosophila model of this disease has been shown to display phenotypes bearing similarity to Fragile X symptoms. Most notably, we previously identified naive courtship and memory deficits in young adults with this model that appear to be due to enhanced metabotropic glutamate receptor (mGluR) signaling. Herein we have examined age-related cognitive decline in the Drosophila Fragile X model and found an age-dependent loss of learning during training. We demonstrate that treatment with mGluR antagonists or lithium can prevent this age-dependent cognitive impairment. We also show that treatment with mGluR antagonists or lithium during development alone displays differential efficacy in its ability to rescue naive courtship, learning during training and memory in aged flies. Furthermore, we show that continuous treatment during aging effectively rescues all of these phenotypes. These results indicate that the Drosophila model recapitulates the age-dependent cognitive decline observed in humans. This places Fragile X in a category with several other diseases that result in age-dependent cognitive decline. This demonstrates a role for the Drosophila Fragile X Mental Retardation Protein (dFMR1) in neuronal physiology with regard to cognition during the aging process. Our results indicate that misregulation of mGluR activity may be causative of this age onset decline and strengthens the possibility that mGluR antagonists and lithium may be potential pharmacologic compounds for counteracting several Fragile X symptoms.


Subject(s)
Aging/psychology , Cognition Disorders/drug therapy , Disease Models, Animal , Animals , Animals, Genetically Modified , Behavior, Animal , Drosophila , Female , Learning , Male , Memory
11.
Fly (Austin) ; 3(1): 39-49, 2009.
Article in English | MEDLINE | ID: mdl-19164936

ABSTRACT

Development of a drug and its safe therapeutic application necessitates using animal models for testing purposes. While testing with mammalian models is essential prior to approval for human trials, the use of invertebrate animal models that are amenable to molecular genetic manipulations provide experimental and biological advantages that can streamline the discovery and testing process. Among the benefits of a genetics-based approach is the ability to screen for genes/proteins that may be novel drug targets, and the expedited development of genetic backgrounds that more accurately reflect a specific disease state. An invertebrate model may provide a more robust phenotype for screening, a situation that may arise when there is unanticipated genetic redundancy present in the mammalian model. Finally, the comparatively short generation time and fecundity of invertebrate models allows for increased experimental throughput. Together, these factors may contribute towards savings in time and cost during the drug discovery process.


Subject(s)
Drosophila/drug effects , Drosophila/genetics , Drug Discovery/methods , Animal Testing Alternatives , Animals , Drosophila/physiology , Drug Evaluation, Preclinical/methods , Female , Genome, Insect , Humans , Male , Models, Animal , Models, Genetic , Models, Neurological , Mutation , Oncogenes , Species Specificity , Transcriptional Activation/drug effects
12.
Behav Brain Res ; 196(2): 220-7, 2009 Jan 23.
Article in English | MEDLINE | ID: mdl-18831990

ABSTRACT

We present a 2-day water maze protocol that addresses some of potential confounds present in the water maze when using the aged subjects typical of studies of neurodegenerative disorders, such as Alzheimer's disease. This protocol is based on an initial series of training trials with a visible platform, followed by a memory test with a hidden platform 24h later. We validated this procedure using aged (15-18m) mice expressing three Alzheimer's disease-related transgenes, PS1(M146 V), APP(Swe), and tau(P301L). We also tested these triple transgenic mice (3xTG) and age and sex-matched wild-type (WT) in a behavioral battery consisting of tests of motor coordination (balance beam), spatial memory (object displacement task) visual acuity (novel object recognition task) and locomotor activity (open field). 3xTG mice had significantly longer escape latencies in the memory trial of the 2-day water maze test than WT and than their own baseline performance in the last visible platform trial. In addition, this protocol had improved sensitivity compared to a typical probe trial, since no significant differences between genotypes were evident in a probe trial conducted 24h after the final training trial. The 2-day procedure also resulted in good reliability between cohorts, and controlled for non-cognitive factors that can confound water maze assessments of memory, such as the significantly lower locomotor activity evident in the 3xTG mice. A further benefit of this method is that large numbers of animals can be tested in a short time.


Subject(s)
Maze Learning/physiology , Swimming/psychology , Alzheimer Disease/genetics , Animals , Female , Genotype , Humans , Male , Memory/physiology , Memory, Short-Term/physiology , Mice , Mice, Transgenic , Motor Activity/physiology , Postural Balance/physiology , Psychomotor Performance/physiology , Recognition, Psychology/physiology , Reproducibility of Results , Sex Characteristics , Space Perception/physiology , Visual Acuity/physiology
13.
Neuron ; 45(5): 753-64, 2005 Mar 03.
Article in English | MEDLINE | ID: mdl-15748850

ABSTRACT

Fragile X syndrome is a leading heritable cause of mental retardation that results from the loss of FMR1 gene function. A Drosophila model for Fragile X syndrome, based on the loss of dfmr1 activity, exhibits phenotypes that bear similarity to Fragile X-related symptoms. Herein, we demonstrate that treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium can rescue courtship and mushroom body defects observed in these flies. Furthermore, we demonstrate that dfmr1 mutants display cognitive deficits in experience-dependent modification of courtship behavior, and treatment with mGluR antagonists or lithium restores these memory defects. These findings implicate enhanced mGluR signaling as the underlying cause of the cognitive, as well as some of the behavioral and neuronal, phenotypes observed in the Drosophila Fragile X model. They also raise the possibility that compounds having similar effects on metabotropic glutamate receptors may ameliorate cognitive and behavioral defects observed in Fragile X patients.


Subject(s)
Courtship , Disease Models, Animal , Fragile X Syndrome/drug therapy , Mushroom Bodies/physiology , Neuronal Plasticity/physiology , Animals , Courtship/psychology , Drosophila , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Amino Acid Antagonists/therapeutic use , Female , Fragile X Syndrome/genetics , Fragile X Syndrome/psychology , Lithium/pharmacology , Lithium/therapeutic use , Male , Memory/drug effects , Memory/physiology , Mushroom Bodies/drug effects , Neuronal Plasticity/drug effects , Synapses/drug effects , Synapses/physiology
14.
J Gen Physiol ; 122(5): 569-81, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14581583

ABSTRACT

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R), a Ca2+-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca2+ signals in many cell types. Three InsP3R isoforms are expressed in different subcellular locations, at variable relative levels with heteromultimer formation in different cell types. A proposed reason for this diversity of InsP3R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca2+ concentrations ([Ca2+]i), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca2+] in mediating high [Ca2+]i inhibition of InsP3R gating by studying single endogenous type 1 InsP3R channels through patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentrations of a calmodulin antagonist nor overexpression of a dominant-negative Ca2+-insensitive mutant calmodulin affected inhibition of gating by high [Ca2+]i. However, a novel, calmodulin-independent regulation of [Ca2+]i inhibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with [Ca2+] approximately 400 nM were inhibited by 290 muM [Ca2+]i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca2+] (<5 nM, for approximately 300 s) before the patch-clamp experiments reversibly relieved Ca2+ inhibition, with channel activities observed in [Ca2+]i up to 1.5 mM. Although InsP3 activates gating by relieving high [Ca2+]i inhibition, it was nevertheless still required to activate channels that lacked high [Ca2+]i inhibition. Our observations suggest that high [Ca2+]i inhibition of InsP3R channel gating is not regulated by calmodulin, whereas it can be disrupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca2+]i inhibition may not be an immutable property of different InsP3R isoforms. Furthermore, these observations support an allosteric model in which Ca2+ inhibition of the InsP3R is mediated by two Ca2+ binding sites, only one of which is sensitive to InsP3.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Calcium/pharmacology , Dose-Response Relationship, Drug , Female , Inositol 1,4,5-Trisphosphate Receptors , Rats , Xenopus laevis
15.
J Gen Physiol ; 122(5): 583-603, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14581584

ABSTRACT

The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po ( approximately 0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2-4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Ion Channel Gating/physiology , Receptors, Cytoplasmic and Nuclear/metabolism , Allosteric Regulation/physiology , Animals , Calcium/pharmacology , Dose-Response Relationship, Drug , Female , Inositol 1,4,5-Trisphosphate Receptors , Ion Channel Gating/drug effects , Membrane Potentials/drug effects , Membrane Potentials/physiology , Rats , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Xenopus laevis
16.
Neuron ; 34(6): 973-84, 2002 Jun 13.
Article in English | MEDLINE | ID: mdl-12086644

ABSTRACT

Fragile X mental retardation is a prominent genetic disorder caused by the lack of the FMR1 gene product, a known RNA binding protein. Specific physiologic pathways regulated by FMR1 function have yet to be identified. Adult dfmr1 (also called dfxr) mutant flies display arrhythmic circadian activity and have erratic patterns of locomotor activity, whereas overexpression of dFMR1 leads to a lengthened period. dfmr1 mutant males also display reduced courtship activity which appears to result from their inability to maintain courtship interest. Molecular analysis fails to reveal any defects in the expression of clock components; however, the CREB output is affected. Morphological analysis of neurons required for normal circadian behavior reveals subtle abnormalities, suggesting that defects in axonal pathfinding or synapse formation may cause the observed behavioral defects.


Subject(s)
Circadian Rhythm/genetics , Courtship , Drosophila Proteins/deficiency , Drosophila Proteins/genetics , Mutation/genetics , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , RNA-Binding Proteins , Alleles , Animals , Circadian Rhythm/physiology , Drosophila , Female , Fragile X Mental Retardation Protein , Insect Proteins/genetics , Insect Proteins/physiology , Male , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/physiology , Nerve Growth Factors/genetics , Nerve Growth Factors/physiology , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/physiology , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Period Circadian Proteins
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