A CHEMOGENETIC EVALUATION OF DELTA SUBUNIT EXCLUSION FROM GABA SYNAPSES

GABA 突触排除 Delta 亚基的化学遗传学评估

• 批准号: 9349595
• 负责人: STEVEN J MENNERICK
• 金额: $ 19.06万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-08 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9349595
• 关键词: Affinity; Amino Acids; Animals; Brain; Breeding; CRISPR/Cas technology; Cause of Death; Cells; Characteristics; Cognition; Cytoplasmic Granules; Data; Epilepsy; Evaluation; Exclusion; GTP-Binding Protein alpha Subunits, Gs; Hippocampus (Brain); Location; Mediating; Modeling; Mood Disorders; Moods; Mus; Mutate; Neurons; Pharmaceutical Preparations; Pharmacology; Phase; Picrotoxin; Play; Point Mutation; Receptor Activation; Regulatory Element; Resistance; Role; Schizophrenia; Signal Transduction; Slice; Specificity; Synapses; Technology; Testing; Therapeutic; Validation; Work; cell type; delta opioid receptor; disability; experimental study; extracellular; gamma-Aminobutyric Acid; induced pluripotent stem cell; innovation; insight; knockin animal; neocortical; neuropsychiatric disorder; positive allosteric modulator; receptor; response; trafficking

δ subunits are subunits of GABAA receptors that are found in receptors that mediate tonic inhibition in several neuronal types. It has been suggested that active exclusion of δ-containing receptors from synapses, by virtue of amino acid residues unique to δ, plays a large role in δ’s preferential participation in tonic currents. Here we investigate the alternative hypothesis that weak expression, rather than active exclusion, limits the role of δ containing receptors to tonic influences. The inability to pharmacologically isolate δ receptors is a large reason that conclusions remain elusive. We investigate the role of δ-containing receptors in synaptic and tonic currents using a chemogenetic approach. We isolate δ contributions by expressing a δ subunit harboring a point mutation that renders receptors resistant to the classical non-competitive GABAA receptor antagonist picrotoxin. We thus study δ contributions in isolation from other classes (primarily γ containing). Preliminary data utilize a heterologous expression model in primary neurons in the context of endogenous subunits. Proposed experiments extend the work to knock-in animals harboring the mutated subunit under the control of endogenous regulators of expression. Results to date support the idea that δ is not actively excluded from synapses. Continued support for this idea would cause the field to re-think active extracellular localization as an explanation for δ’s preferential role in tonic currents. We believe it is likely that low-level GABA acting on δ low abundance, high-affinity receptors throughout the cell, are sufficient to account for the preferential role that δ plays in tonic currents.

δ亚基是GABAA受体的亚基，其存在于介导几种神经元类型中的紧张性抑制的受体中。有人认为，由于δ特有的氨基酸残基，含有δ的受体从突触中主动排斥，在δ优先参与紧张性电流中起着重要作用。在这里，我们调查的替代假设，弱表达，而不是主动排斥，限制了δ含有受体的作用，紧张的影响。无法分离δ受体是结论仍然难以捉摸的一个重要原因。我们使用化学发生学方法研究了含δ受体在突触电流和强直电流中的作用。我们通过表达携带点突变的δ亚基来分离δ贡献，该点突变使受体对经典的非竞争性GABAA受体拮抗剂印防己毒素具有抗性。因此，我们将δ贡献与其他类别（主要是含γ的）隔离开来研究。初步数据利用内源性亚基背景下的原代神经元中的异源表达模型。拟议的实验将工作扩展到在内源性表达调节剂的控制下携带突变亚基的敲入动物。迄今为止的结果支持δ并没有被积极地排除在突触之外的观点。继续支持这一观点将导致该领域重新考虑活性细胞外定位作为δ在紧张性电流中的优先作用的解释。我们认为，低水平的GABA作用于整个细胞中δ低丰度、高亲和力的受体可能就足够了 来解释δ在紧张电流中的优先作用。