Modulation Of Neuronal Channels and Receptors in the Brain

大脑神经元通道和受体的调节

• 批准号: 7593975
• 负责人: JERREL L YAKEL
• 金额: $ 159.54万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593975
• 关键词: Acetylcholine; Agonist; Alanine; Alleles; Alzheimer' s Disease; Amino Acid Substitution; Amino Acids; Amygdaloid structure; Animals; Apolipoprotein E; Area; Arginine; Binding; Binding Sites; Brain; Brain Injuries; Brain region; Calcium; Calcium Signaling; Cells; Charge; Cholinergic Agents; Chromosome Pairing; Chronic; Cognitive; Cognitive deficits; Communication; Complex; Condition; Corpus striatum structure; Cysteine; DNA Sequence Rearrangement; Data; Dementia; Dendrites; Development; Diffuse; Docking; Dopamine; Dose; Electrophysiology (science); Emotional; Epilepsy; Exposure to; Fright; Functional disorder; Fura-2; Gated Ion Channel; Hippocampus (Brain); Human; Hydrophobic Interactions; Imaging Techniques; Impaired cognition; Insecticides; Interneurons; Investigation; Ion Channel Gating; Kinetics; Lead; Ligands; Link; Localized; Location; Maps; Mediating; Memory; Modeling; Molecular; Motor; Mutate; Mutation; Nature; Nervous system structure; Neurodegenerative Disorders; Neurons; Nicotine; Nicotinic Receptors; Parkinson Disease; Patients; Peptide Fragments; Peptides; Performance; Phenylalanine; Physiological; Physiological Processes; Play; Positioning Attribute; Postsynaptic Membrane; Pregnant Women; Presynaptic Terminals; Process; Rate; Rattus; Recovery; Regulation; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Role; Rotenone; Schizophrenia; Signal Transduction; Site; Slice; Smoke; Structural Models; Structure; Substantia nigra structure; Sulfhydryl Reagents; Symptoms; Synapses; Synaptic Cleft; Synaptic Transmission; Synaptic plasticity; Techniques; Testing; Therapeutic; Tobacco Dependence; Torpedo; Tryptophan; Tyrosine; Valine; Xenopus oocyte; alpha Bungarotoxin; basal forebrain; base; cholinergic; desensitization; disorder risk; dopaminergic neuron; environmental agent; genetic risk factor; improved; in utero; insight; memory process; mind control; nerve supply; nervous system disorder; neuronal cell body; neurotransmitter release; patch clamp; protein aminoacid sequence; receptor; receptor function; response; spatiotemporal; three-dimensional modeling; voltage; voltage clamp; Acetylcholine; Agonist; Alanine; Alleles; Alzheimer' s Disease; Amino Acid Substitution; Amino Acids; Amygdaloid structure; Animals; Apolipoprotein E; Area; Arginine; Binding; Binding Sites; Brain; Brain Injuries; Brain region; Calcium; Calcium Signaling; Cells; Charge; Cholinergic Agents; Chromosome Pairing; Chronic; Cognitive; Cognitive deficits; Communication; Complex; Condition; Corpus striatum structure; Cysteine; DNA Sequence Rearrangement; Data; Dementia; Dendrites; Development; Diffuse; Docking; Dopamine; Dose; Electrophysiology (science); Emotional; Epilepsy; Exposure to; Fright; Functional disorder; Fura-2; Gated Ion Channel; Hippocampus (Brain); Human; Hydrophobic Interactions; Imaging Techniques; Impaired cognition; Insecticides; Interneurons; Investigation; Ion Channel Gating; Kinetics; Lead; Ligands; Link; Localized; Location; Maps; Mediating; Memory; Modeling; Molecular; Motor; Mutate; Mutation; Nature; Nervous system structure; Neurodegenerative Disorders; Neurons; Nicotine; Nicotinic Receptors; Parkinson Disease; Patients; Peptide Fragments; Peptides; Performance; Phenylalanine; Physiological; Physiological Processes; Play; Positioning Attribute; Postsynaptic Membrane; Pregnant Women; Presynaptic Terminals; Process; Rate; Rattus; Recovery; Regulation; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Role; Rotenone; Schizophrenia; Signal Transduction; Site; Slice; Smoke; Structural Models; Structure; Substantia nigra structure; Sulfhydryl Reagents; Symptoms; Synapses; Synaptic Cleft; Synaptic Transmission; Synaptic plasticity; Techniques; Testing; Therapeutic; Tobacco Dependence; Torpedo; Tryptophan; Tyrosine; Valine; Xenopus oocyte; alpha Bungarotoxin; basal forebrain; base; cholinergic; desensitization; disorder risk; dopaminergic neuron; environmental agent; genetic risk factor; improved; in utero; insight; memory process; mind control; nerve supply; nervous system disorder; neuronal cell body; neurotransmitter release; patch clamp; protein aminoacid sequence; receptor; receptor function; response; spatiotemporal; three-dimensional modeling; voltage; voltage clamp

There have been several major accomplishments within the past fiscal year. The first is that we have discovered the presence of functional nAChR-mediated current responses from rat amygdala. The amygdala complex, the limbic structure important for emotional memory formation, receives cholinergic innervation from the basal forebrain. Although cholinergic drugs have been shown to regulate passive avoidance performance via the amygdala, the neuronal subtypes and circuits involved in this regulation are unknown. Our results suggest possible mechanisms whereby nAChRs may be playing a significant role in fear and aversively-motivated memory. Second, we utilized highly localized and rapid iontophoretic agonist delivery, combined with patch-clamp electrophysiology recordings and conventional fura-2 fluorescent imaging techniques, to compare the intracellular calcium levels in the soma and dendrites of rat hippocampal CA1 interneurons in the slice due to the activation of these alpha7 nAChRs. We found that in the dendrites, whereas the amplitudes of the current responses were smaller and the decay kinetics faster than the responses in the soma, the amplitudes of the calcium signals were significantly larger. Cultured hippocampal neurons were studied since the dendritic field lies in the same focal plane, which allowed for a broader investigation of the spatiotemporal dynamics of calcium signalling. In cultured neurons, the calcium signals in the dendrites were similar to those in slices. Interestingly in cultures, even though the amplitude of the alpha7 nAChR-mediated currents dramatically decreased with distance from the soma (from approximately 20-250 microm), the amplitude of the calcium signals did not correlate with distance. This indicates that the relative efficacy of alpha7 nAChR activation to increase calcium levels in dendrites increased severalfold with distance from the soma. These results may have implications for the role that alpha7 nAChRs have in regulating various signal transduction cascades, synaptic plasticity, and memory processes, via significant changes in calcium levels. Third, we have continued to study the possible involvement of nAChRs relating to Alzheimers disease (AD) since dysfunctions in these receptors have been linked to AD, a human neurological disorder that is the leading cause of dementia. The most significant genetic risk factor for the development of Alzheimers disease is inheritance of the E4 allele of apolipoprotein E (apoE). Recent data has demonstrated the ability of apoE-derived peptides to inhibit nAChRs in rat hippocampus. We investigated the functional interaction between nAChRs and apoE-derived peptides in Xenopus oocytes expressing selected nAChRs. Both a 17 amino acid peptide fragment, apoE133-149, and an 8 amino acid peptide, apoE141-148, were able to maximally block ACh-mediated peak current responses for homomeric alpha7 nAChRs. ApoE peptide inhibition was dose-dependent, and voltage- and activity-independent. The current findings suggest that apoE peptides are non-competitive for acetylcholine and do not block functional alpha-bungarotoxin binding. Amino acid substitutions in the apoE peptide sequence suggest that the arginines are critical for peptide blockade of the alpha7 nAChR. Furthermore, mutation of tryptophan (W) 55 to alanine on the alpha7 nAChR blocked apoE peptide-induced inhibition of ACh-mediated alpha7 nAChR responses. Additional mutations at W55 suggest that hydrophobic interactions between the receptor and apoE141-148 are key for inhibition of alpha7 nAChR function. A mutated apoE peptide also demonstrated decreased inhibition at alpha7-W55A nAChRs as well as activity-dependent inhibition of both wildtype alpha7 nAChRs and alpha7-W55A receptors. Finally, a three-dimensional model of the alpha7 nAChR was developed based on the recently refined Torpedo nAChR. A structural model was proposed for the binding of apoE141-148 to the alpha7 nAChR where the peptide binds at the interface between two subunits, near the ACh binding site. Similar to the functional data, the computational docking suggests the importance of hydrophobic interactions between the alpha7 nAChR and the apoE peptide for inhibition of receptor function. The current data suggest a mode for apoE peptide binding that directly blocks alpha7 nAChR activity, and consequently may disrupt nAChR signaling. Interestingly, the alpha7-W55A nAChR desensitized more slowly, and recovered from desensitization more rapidly, than wildtype alpha7 nAChRs. This interpretation was validated by kinetic modeling of receptor function. Mutating W55 to other aromatic residues (phenylalanine or tyrosine) had no significant effect on the kinetics of desensitization, whereas mutation to various hydrophobic residues (alanine, cysteine, or valine) significantly decreased the rate of onset and increased the rate of recovery from desensitization. To gain insight into possible structural rearrangements during desensitization, we probed the accessibility of W55 by mutating W55 to cysteine (alpha7-W55C) and testing the ability of various sulfhydryl reagents to react with this cysteine. Several charged (both positively and negatively) sulfhydryl reagents blocked ACh-induced responses for alpha7-W55C nAChRs, whereas a neutral sulfhydryl reagent potentiated responses. These data suggest that W55 plays an important role in both the onset and recovery from desensitization in the rat alpha7 nAChR, and that aromatic residues at position 55 are critical for maintaining rapid desensitization. Furthermore, these data suggest that W55 may be a potential target for modulatory agents operating via hydrophobic interactions.

在过去的财政年度中，已经取得了一些重大成就。 首先是我们发现了大鼠杏仁核的功能性NACHR介导的电流反应。 杏仁核复合体是对情绪记忆形成重要的边界结构，它从基础前脑接收胆碱能神经。 尽管已证明胆碱能药物通过杏仁核调节被动回避性能，但该调节中涉及的神经元亚型和电路尚不清楚。 我们的结果表明，NACHR可能在恐惧和厌恶动机的记忆中发挥重要作用。 其次，我们利用了高度局部和快速的离子电遗传激动剂的传递，结合了贴片钳电生理记录和常规的FURA-2荧光成像技术，以比较soma的细胞内钙水平和大鼠海马CA1 Interneurons在Slice中，由于这些Alpha的活性nach nACh n chan n chan slice slice slice slice slice。 我们发现，在树突中，当前反应的幅度幅度较小，并且衰减动力学比SOMA的响应更快，而钙信号的幅度明显更大。研究了培养的海马神经元，因为树突状场位于同一焦平面，这可以对钙信号的时空动力学进行更广泛的研究。在培养的神经元中，树突中的钙信号与切片中的钙信号相似。有趣的是，在培养物中，即使Alpha7 NACHR介导的电流的幅度随着距离SOMA的距离（大约20-250 microm）的距离急剧下降，但钙信号的振幅与距离无关。这表明α7NACHR激活增加树突中钙水平的相对功效增加了几倍，距离soma距离。这些结果可能对Alpha7 NACHR在调节各种信号转导级联，突触可塑性和记忆过程中的作用有影响，这可能会通过钙水平的重大变化。 第三，我们继续研究与阿尔茨海默氏病（AD）有关的NACHR的可能参与，因为这些受体的功能障碍与AD有关，AD是一种人类神经系统疾病，这是痴呆症的主要原因。 阿尔茨海默氏病发展的最重要的遗传危险因素是载脂蛋白E（APOE）E4等位基因的遗传。最近的数据表明，APOE衍生的肽在大鼠海马中抑制NACHR的能力。 我们研究了表达选定NACHR的爪蟾卵母细胞中NACHR和APOE衍生的肽之间的功能相互作用。 17个氨基酸肽片段APOE133-149和8个氨基酸肽APOE141-148都能够最大程度地阻断同源α7NACHR的ACH介导的峰值电流响应。 APOE肽抑制是剂量依赖性的，并且无独立的电压和活性。当前的发现表明，APOE肽对乙酰胆碱是非竞争力的，并且不会阻止功能性α-bungarotoxin结合。 APOE肽序列中的氨基酸取代表明，精氨酸对于α7NACHR的肽阻塞至关重要。 此外，在α7NACHR上，色氨酸（W）55对丙氨酸的突变阻断了APOE肽诱导的抑制ACH介导的α7NACHR响应。 W55处的其他突变表明受体与APOE141-148之间的疏水相互作用是抑制alpha7 NACHR功能的关键。突变的APOE肽还表明，α7-W55A NACHRS的抑制作用降低，以及对Wildtype alpha7 NACHR和α7-W55A受体的活性依赖性抑制作用。最后，基于最近精制的鱼雷NACHR开发了Alpha7 NACHR的三维模型。提出了一个结构模型，用于将APOE141-148与Alpha7 NACHR结合，其中肽在ACH结合位点附近的两个亚基之间的界面上结合。与功能数据相似，计算对接表明α7NACHR和APOE肽之间疏水相互作用的重要性以抑制受体功能。当前数据提出了一种直接阻止alpha7 nACHR活性的APOE肽结合的模式，因此可能会破坏NACHR信号传导。 有趣的是，α7-W55A NACHR脱敏的脱敏性更慢，并且比野生型alpha7 nachrs更快地从脱敏中恢复。 通过受体功能的动力学建模来验证这种解释。 将W55突变为其他芳族残基（苯丙氨酸或酪氨酸）对脱敏动力学没有显着影响，而对各种疏水残基（丙氨酸，半胱氨酸或缬氨酸）的突变显着降低了发作速率，并增加了从脱敏中恢复的发病率。 为了深入了解在脱敏过程中可能的结构重排，我们通过将W55突变为半胱氨酸（alpha7-W55C）并测试各种硫磺酶与这种半胱氨酸反应的能力来探测W55的可及性。 几种带电的（阳性和负）磺胺试剂阻止了Alpha7-W55C NACHR的ACH诱导的响应，而中性硫酰亚基试剂的反应增强了反应。 这些数据表明，W55在大鼠α7NACHR的脱敏开始和恢复中都起着重要作用，而位置55处的芳族残基对于维持快速脱敏至关重要。 此外，这些数据表明W55可能是通过疏水相互作用运行的调节剂的潜在目标。