6 results on '"Christopher E. Bope"'
Search Results
2. Analyzing the mechanisms that facilitate the subtype-specific assembly of γ-aminobutyric acid type A receptors
- Author
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Catherine Choi, Joshua L. Smalley, Abigail H. S. Lemons, Qiu Ren, Christopher E. Bope, Jake S. Dengler, Paul A. Davies, and Stephen J. Moss
- Subjects
trafficking ,subunit composition ,phosphorylation ,GABAA receptors ,protein purification ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABAARs), which mediate phasic and tonic inhibition, respectively. These two GABAAR subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABAARs contain the α1–3 subunits whereas extrasynaptic GABAARs contain the α4–6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABAAR subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABAARs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABAARs and interact with distinct sets of binding proteins. We also discovered that the β3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABAARs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABAARs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABAARs, and thus the efficacy of neuronal inhibition.
- Published
- 2022
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3. Isolation and Characterization of Multi-Protein Complexes Enriched in the K-Cl Co-transporter 2 From Brain Plasma Membranes
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Joshua L. Smalley, Georgina Kontou, Catherine Choi, Qiu Ren, David Albrecht, Krithika Abiraman, Miguel A. Rodriguez Santos, Christopher E. Bope, Tarek Z. Deeb, Paul A. Davies, Nicholas J. Brandon, and Stephen J. Moss
- Subjects
potassium chloride cotransporter 2 ,proteome ,interactome ,phosphorylation ,protein purification ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.
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- 2020
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4. Spectrin-beta 2 facilitates the selective accumulation of GABA
- Author
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Joshua L, Smalley, Noell, Cho, Shu Fun Josephine, Ng, Catherine, Choi, Abigail H S, Lemons, Saad, Chaudry, Christopher E, Bope, Jake S, Dengler, Chuansheng, Zhang, Matthew N, Rasband, Paul A, Davies, and Stephen J, Moss
- Abstract
Fast synaptic inhibition is dependent on targeting specific GABA
- Published
- 2022
5. The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products
- Author
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Nicholas J. Brandon, Georgina Kontou, Paul Davies, Joshua L. Smalley, David Albrecht, Miguel A. Rodriguez Santos, Tarek Z. Deeb, Stephen J. Moss, Krithika Abiraman, Catherine Choi, Qiu Ren, and Christopher E. Bope
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0303 health sciences ,Transporter ,Biology ,SPTAN1 ,medicine.disease ,FMR1 ,03 medical and health sciences ,Epilepsy ,0302 clinical medicine ,Autism spectrum disorder ,Knockout mouse ,medicine ,Autism ,Neuroscience ,Gene ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
KCC2 plays a critical role in determining the efficacy of synaptic inhibition and deficits in its activity lead to epilepsy and neurodevelopmental delay. Here we use unbiased proteomic analyses to demonstrate that KCC2 forms stable protein complexes in the neuronal plasma membrane with 96 autism and/or epilepsy risk gene (ASD/Epi) products including ANKB, ANKG, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1, and SPTBN1. Many of these proteins are also targets of Fragile-X mental retardation protein (FMRP), the inactivation of which is the leading monogenic cause of autism. Accordingly, the expression of a subset of these KCC2-binding partners was decreased in Fmr1 knockout mice. Fmr1 knockout compromised KCC2 phosphorylation, a key regulatory mechanism for transporter activity and the postnatal development of GABAergic inhibition. Thus, KCC2 is a point of convergence for multiple ASD/Epi risk genes and therapies targeting this transporter may have broad utility in alleviating these heterogeneous disorders and their associated epilepsies.
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- 2020
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6. KCC2 is required for the survival of mature neurons but not for their development
- Author
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Shu Fun Josephine Ng, Jack H. Howden, Christopher E. Bope, Miguel A. Rodriguez Santos, Jake S. Dengler, Matt R. Kelley, Qiu Ren, Paul Davies, Ross A. Cardarelli, Catherine Choi, Josef T. Kittler, Joshua L. Smalley, Stephen J. Moss, Georgina Kontou, and Nicholas J. Brandon
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Male ,0301 basic medicine ,Programmed cell death ,Neurogenesis ,KCC2 ,Poly ADP ribose polymerase ,Primary Cell Culture ,TLE, temporal lobe epilepsy ,Apoptosis ,Biochemistry ,Epileptogenesis ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,AAV, adeno-associated virus ,Chlorides ,Receptors, GABA ,Seizures ,Animals ,TTX, tetrodotoxin ,Receptor ,Molecular Biology ,gamma-Aminobutyric Acid ,GFP, green fluorescent protein ,Mice, Knockout ,Neurons ,KO, knockout ,Symporters ,030102 biochemistry & molecular biology ,PARP, poly (ADP-ribose) polymerase (PARP) ,Cell Biology ,KCC2, K+/Cl− cotransporter 2 ,DIV, days in vitro ,Cell biology ,Mice, Inbred C57BL ,cell death ,030104 developmental biology ,nervous system ,chemistry ,Potassium ,Tetrodotoxin ,Female ,extrinsic pathway ,Cotransporter ,GABAAR, γ-aminobutyric acid type A receptors ,Intracellular ,Research Article - Abstract
The K+/Cl− cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl− levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.
- Published
- 2021
- Full Text
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