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: 8746478
• 负责人: Stephen R Ikeda
• 金额: $ 157.61万
• 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/8746478
• 关键词: 2-arachidonylglycerol; Acetates; Address; Adenylate Cyclase; Adipose tissue; Agonist; Alcohol consumption; Alcoholism; Animal Model; Anti-Obesity Agents; Biochemical; Biological; Biological Assay; Biological Models; Biosensor; Blood; Calcium; Calcium Channel; Calcium Channel Inhibition; Cardiovascular system; Cell membrane; Cells; Collaborations; Coupled; Coupling; Data; Development; Diabetic Ketoacidosis; Endocannabinoids; Ethanol; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Future; G protein-coupled inwardly-rectifying potassium channel; G-Protein-Coupled Receptors; GTP-Binding Proteins; Ganglia; Gated Ion Channel; Genetic; Genetic Polymorphism; Genetic Techniques; Glycine; Goals; Hemagglutinin; Heterotrimeric GTP-Binding Proteins; Human; Imaging Techniques; Investigation; Ion Channel; Ketone Bodies; Laboratories; Life; Ligands; Measurement; Mediating; Mesentery; Metabolism; Methodology; Molecular; N-Type Calcium Channels; Nervous system structure; Neural Pathways; Neurons; Optics; Pathway interactions; Peripheral; Physiological; Plasma; Play; Predisposition; Primates; Property; Propionates; Protein Family; Proteins; Publishing; RBM5 gene; Rattus; Reporting; Research; Rodent; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Sodium Channel; Spinal Ganglia; Staging; Staining method; Stains; Sympathetic Ganglia; Symptoms; System; Techniques; Testing; Volatile Fatty Acids; Zebrafish; abn-cbd; addiction; alcohol response; alcohol reward; analog; anandamide; base; beta-Hydroxybutyrate; design; electrical property; feeding; hangover; ketogenic diet; luminescence; molecular imaging; mutant; novel; 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, completed 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. The published findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies utilizing this model system. 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 was completed that examined a putative endogenous ligand for the G-protein coupled receptor GPR18. Recent studies proposed that N-arachidonyl glycine (NAGly), a carboxylic analog of anandamide, was 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. (2013) N-arachidonyl glycine (NAGly does not activate G protein-coupled receptor 18 (GPR18) signaling via canonical pathways. Mol Pharmacol 83:267-82. A third project 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 acids (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. Finally, our data indicate that a ketone body, beta-hydroxybutyrate, acts as an agonist at this receptor. These findings are potentially important for several reasons. First, GPR41 may be expressed in neurons that innervate adipose tissue and thus be an important target for anti-obesity drugs. Second, we proposed that beta-hydroxybutyrate may serve as the primary endogenous ligand for sympathetic ganglia. This finding has implications for conditions in which plasma beta hydroxybutyrate is elevated such as diabetic ketoacidosis or ketogenic diets. Third, 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 and primate-specific segmental duplication GPR42 contribute to susceptibility to alcoholism in humans. Fourth, 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. Won Y-J, Lu, VB, Puhl HL, Ikeda SR. (2013) Beta-hydroxybutyrate modulates N-type calcium channels in rat sympathetic neurons by acting as an agonist for the G protein coupled receptor FFA3 (GPR41). In revision.

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开发斑马鱼神经元系统，适用于在开发阶段研究钠和钙通道调节和功能，该开发阶段利用了该模型有机体的遗传性锻造性和光学透明度。已发表的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，因此为利用该模型系统的未来研究提供了基础。 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。 第二项研究完成了G蛋白偶联受体GPR18的假定内源配体。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。 （2013）N-弧菌甘氨酸（Nagly不会激活G蛋白偶联受体18（GPR18）信号通过规范途径传导。MOLPHORMACOL 83：267-82。 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“去甲化” 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。最后，我们的数据表明，酮体β-羟基丁酸酯在该受体中充当激动剂。这些发现可能有几个原因很重要。首先，GPR41可以在神经化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，我们提出β-羟基丁酸酯可以作为交感神经的主要内源配体。这一发现对血浆β羟基丁酸的条件具有影响，例如糖尿病性酮症酸中毒或生酮饮食。第三，乙酸是乙醇的主要代谢产物。因此，GPR41可能参与对乙醇（尤其是宿醉症状）和成瘾的反应。 GPR41和灵长类动物特异性的分段重复GPR42中的多态性可能导致人类酒精中毒的易感性。第四，由于GPR41可能能够通过血液乙酸水平间接检测乙醇，因此可能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。 Won Y-J，Lu，VB，Puhl HL，Ikeda Sr。 （2013）β-羟基丁酸酯通过充当G蛋白偶联受体FFA3（GPR41）的激动剂，调节大鼠交感神经元中的N型钙通道。在修订中。