Neuronal Ion Channel Modulation By Second Messengers

第二信使对神经元离子通道的调节

• 批准号: 8559270
• 负责人: Stephen R Ikeda
• 金额: $ 142.89万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 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/8559270
• 关键词: 2-arachidonylglycerol; Acetates; Address; Adenylate Cyclase; Adipose tissue; Adolescent; Afferent Neurons; Agonist; Alcohol consumption; Alcoholism; Animal Model; Anti-Obesity Agents; Axon; Biochemical; Biological; Biological Assay; Biological Models; Biosensor; Blood; Calcium; Calcium Channel; Calcium Channel Inhibition; Cell membrane; Cells; Collaborations; Coupled; Coupling; Development; Endocannabinoids; Ethanol; Family; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Future; G protein-coupled inwardly-rectifying potassium channel; G-Protein Signaling Pathway; G-Protein-Coupled Receptors; GTP-Binding Proteins; Ganglia; Gated Ion Channel; Genetic; Genetic Polymorphism; Genetic Techniques; Glycine; Goals; Green Fluorescent Proteins; Hemagglutinin; Heterotrimeric GTP-Binding Proteins; Human; Imaging Techniques; Investigation; Ion Channel; Laboratories; Life; Ligands; Manuscripts; Measurement; Mediating; Mesentery; Methodology; Minor; Modality; Molecular; N-Type Calcium Channels; Nervous system structure; Neural Pathways; Neurons; Neurotransmitters; Optics; Pathway interactions; Peripheral; Phase; Physiological; Play; Predisposition; Preparation; Property; Propionates; Protein Family; Proteins; RBM5 gene; Rattus; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Second Messenger Systems; Sensory; Signal Pathway; Signal Transduction; Sodium; Sodium Channel; Spinal Ganglia; Staging; Staining method; Stains; Symptoms; System; Techniques; Testing; Tetrodotoxin; Vertebrates; Volatile Fatty Acids; Zebrafish; abn-cbd; addiction; alcohol response; alcohol reward; analog; anandamide; base; design; electrical property; hangover; luminescence; molecular imaging; mutant; neuronal cell body; novel; omega-Conotoxins; patch clamp; promoter; protein activation; ras-Related G-Proteins; receptor; receptor expression; response; second messenger; trafficking; voltage

The primary focus of the section is to further our understanding of the molecular basis of signaling between G protein coupled receptors and voltage gated ion channels in neurons using electrophysiological, molecular, and imaging techniques. A project, done in collaboration with Dr. Fumihito Ono, resulted in the development of a zebrafish neuronal system suitable for investigating sodium and calcium channel modulation and function during a development stage that leverages the genetic malleability and optical transparency of this model organism. Dorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available. We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven green fluorescent protein were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch- clamp studies. Two kinetically distinct tetrodotoxin-sensitive sodium currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating sodium currents were preferentially expressed in relatively large neurons, while slowly-inactivating sodium currents was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab, and zscn5Laa are possible candidates for these sodium current components. Voltage-gated calcium currents were primarily comprised of a high-voltage activated component arising from omega-conotoxin GVIA-sensitive CaV2.2 (N-type) calcium channels. A few neurons displayed a minor low-voltage-activated component. Calcium currents in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability. Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies. Won YJ, Ono F, Ikeda SR. Characterization of Na+ and Ca2+ channels in zebrafish dorsal root ganglion neurons. PLoS One. 2012;7(8):e42602. A second study examined an endogenous ligand for the G-protein coupled receptor GPR18. Recent studies propose that N-arachidonyl glycine (NAGly), a carboxylic analog of anandamide, is an endogenous ligand of GPR18. However, other studies failed to detect activation of GPR18 by NAGly. To address this inconsistency, we investigated GPR18 coupling in a native neuronal system with endogenous signaling pathways and effectors. We heterologously expressed GPR18 in rat sympathetic neurons and examined the modulation of N-type calcium channels. Proper expression and trafficking of receptor was confirmed by the rim-like fluorescence of fluorescently-tagged receptor and the positive staining of external hemagglutinin-tagged GPR18-expressing cells. Application of NAGly on GPR18-expressing neurons did not inhibit calcium currents, but instead potentiated currents in a voltage-dependent manner, similar to what has previously been reported by our laboratory (Guo et al., 2008; J Neurophysiol, 100:1147). Other proposed agonists of GPR18, including anandamide and abnormal cannabidiol, also failed to induce inhibition of calcium currents. Mutants of GPR18, designed to constitutively activate receptors, did not tonically inhibit calcium currents indicating a lack of GPR18 activation or coupling to endogenous G proteins. Other downstream effectors, G protein-coupled inwardly-rectifying potassium channels and adenylate cyclase, were not modulated by GPR18 signaling. Furthermore, GPR18 did not couple pathway utilizing to Gs, Gz, or G15. These results argue that NAGly is not an agonist for GPR18 or that GPR18 signaling involves non-canonical pathways not examined in these studies. Lu, VB, Puhl, HL, Ikeda, SR. N-arachidonyl glycine (NAGly does not activate G protein-coupled receptor 18 (GPR18) signaling via canonical pathways. Manuscript in revision. A third project, currently nearing first phase completion involves the investigation of a recently "de-orphanized" G-protein coupled receptor termed GPR41 or FFAR3 in rodent sympathetic neurons. These receptors use short-chained fatty acid (SCFA; e.g., acetate and propionate) as endogenous ligands but little else is known. We have discovered that GPR41 is natively expressed and functionally coupled to calcium channels in sympathetic neurons (primarily the celiac/superior mesenteric ganglia). We have also generated evidence that receptor expression is highest (and possibly functional confined to) paravertebral and prevertebral sympathetic neurons. These findings are potentially important for several reasons. First, GPR41 maybe expressed in neurons that innervate adipose tissue and thus be an important target for anti-obesity drugs. Second, acetate is a major metabolite of ethanol. Hence, GPR41 may be involved in both the response to ethanol (especially hangover symptoms) and addiction. It is possible that polymorphisms in GPR41 contribute to susceptibility to alcoholism in humans. Third, as GPR41 is likely capable of detecting ethanol indirectly via blood acetate levels, it may be possible to leverage this property to interrupt neural pathways contributing to addiction in model organisms using genetic techniques.

本节的主要重点是进一步了解G蛋白耦合受体和电压门控离子通道之间使用电生理，分子和成像技术中信号传导的分子基础的理解。 一个与Fumihito Ono博士合作完成的项目，导致了斑马鱼神经元系统的开发，适用于在开发阶段研究钠和钙通道调节和功能，该阶段利用了该模型有机体的遗传性延展性和光学透明度。啮齿动物的背根神经节（DRG）somata提供了一个出色的模型系统，可以使用电生理研究研究离子通道性能和调节。与其他脊椎动物一样，斑马鱼（Danio rerio）DRG是分段组织的，并具有分叉在每个体段中的外围轴突。然而，与哺乳动物的同伴相比，斑马鱼DRG感觉神经元的电性能相对尚未探索，因为尚未提供适合电生理研究的制剂。我们显示了来自少年表达ISL2B启动器驱动的绿色荧光蛋白的酶解离的DRG神经元，可轻松鉴定出荧光显微镜，并且可与常规的全细胞斑块夹研究。发现了两种动力学上不同的四毒素敏感钠电流（快速和缓慢灭活）。在相对较大的神经元中优先表达迅速吸收的钠电流，而在较小的DRG神经元中，缓慢灭活的钠电流更为普遍。 RT-PCR分析表明ZSCN1AA/AB，ZSCN8AA/AB，ZSCN4AB和ZSCN5LAA可能是这些钠电流成分的候选者。电压门控钙电流主要由由欧米茄毒素GVIA敏感CAV2.2（N型）钙通道产生的高压活化成分组成。一些神经元显示出较小的低压激活成分。斑马鱼DRG神经元中的钙电流通过神经递质依赖性或非依赖性的G蛋白信号传导途径调节，具有较大的细胞对细胞响应可变性。我们目前的结果表明，与较高的脊椎动物一样，斑马鱼DRG神经元是由功能上不同的亚群组成的，可能与不同的感觉方式相关。这些发现提供了斑马鱼和啮齿动物DRG神经元电性能的首次比较，因此为将来的研究提供了基础。 Won YJ，Ono F，Ikeda Sr。斑马鱼背根神经元中Na+和Ca2+通道的表征。 PLOS一个。 2012; 7（8）：E42602。 第二项研究检查了G蛋白偶联受体GPR18的内源配体。最近的研究表明，N-弧菌甘氨酸（Nagly）是Anandamide的羧基类似物，是GPR18的内源性配体。但是，其他研究未能检测到Nagly对GPR18的激活。为了解决这种不一致，我们研究了具有内源信号通路和效应子的天然神经元系统中的GPR18耦合。我们在大鼠交感神经元中异源表达GPR18，并检查了N型钙通道的调节。通过荧光受体的边缘样荧光以及外部血凝素标记的GPR18表达细胞的正染色证实了受体的正确表达和运输。 Nagly在表达GPR18的神经元上的应用不会抑制钙电流，而是以电压依赖性的方式抑制电流，类似于我们实验室以前报道的电流（Guo等，2008; J Neurophysiol，100：1147）。 GPR18的其他提议的激动剂，包括anandamide和异常大麻二酚，也未能诱导钙电流的抑制作用。旨在组成性激活受体的GPR18的突变体并未在语调上抑制钙电流，表明缺乏GPR18激活或与内源性G蛋白偶联。其他下游效应子，GPR18信号传导调节了GPR18信号的G蛋白偶联的钾通道和腺苷酸环化酶。此外，GPR18并未将使用GS，GZ或G15的途径磨合。这些结果表明，Nagly不是GPR18的激动剂，或者GPR18信号传导涉及在这些研究中未检查的非规范途径。 lu，vb，puhl，hl，ikeda，sr。 N-蛛网膜甘氨酸（Nagly不会激活G蛋白偶联受体18（GPR18）信号通过规范途径。修订中的手稿。 目前接近第一阶段完成的第三个项目涉及对啮齿动物交感神经元中最近称为GPR41或FFAR3的最近“脱氧化” G蛋白偶联受体的研究。这些受体使用短链的脂肪酸（SCFA；例如乙酸和丙酸酯）作为内源配体，但鲜为人知。我们已经发现，GPR41本地表达，并在功能上耦合到交感神经元（主要是腹腔/肠系膜上神经节）中的钙通道。我们还产生了证据，表明受体表达最高（可能局限于）椎间盘和脊椎前交感神经元。这些发现可能有几个原因很重要。首先，GPR41可能在神经支配脂肪组织的神经元中表达，因此成为抗肥胖药物的重要靶标。其次，乙酸是乙醇的主要代谢物。因此，GPR41可能参与对乙醇（尤其是宿醉症状）和成瘾的反应。 GPR41中的多态性可能导致对人类酒精中毒的易感性。第三，由于GPR41可能能够通过血液乙酸水平间接检测乙醇，因此有可能利用该特性来中断使用遗传技术在模型生物中导致成瘾的神经途径。