Targeted mutagenesis to elucidate the function of understudied ion channels in the central nervous system

定向诱变阐明中枢神经系统中待研究离子通道的功能

• 批准号: 10045757
• 负责人: Marta E Soden
• 金额: $ 15.55万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2022-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10045757
• 关键词: Action Potentials; Address; Adult; Affect; Anxiety; Attention deficit hyperactivity disorder; Automobile Driving; Behavior; Behavioral; Biological Models; CRISPR/Cas technology; Calcium Channel; Cell Separation; Cell membrane; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complex; Cues; Cytomegalovirus; DNA Sequence Alteration; DNA sequencing; Data; Development; Dopamine; Electrophysiology (science); Emotional; Environment; Enzymes; Exhibits; Frequencies; Gene Mutation; Genes; Genome; Goals; Guide RNA; Individual; Injections; Investigation; Ion Channel; Ions; Kinetics; Knock-out; Learning; Light; Link; Measures; Membrane Potentials; Mental disorders; Methods; Modeling; Motivation; Mus; Mutagenesis; Mutation; Neuraxis; Neurons; Neurotransmitters; Nonsense Codon; Nucleus Accumbens; Pattern; Periodicity; Pharmacology; Physiological; Physiology; Play; Polyribosomes; Potassium Channel; Precision therapeutics; Property; Proteins; Research; Rest; Rewards; Role; Scanning; Schizophrenia; Shaker potassium channel; Shapes; Slice; Specificity; Stimulus; Synapses; Synaptic Vesicles; System; Technology; Testing; Therapeutic; Time; Translating; Ventral Tegmental Area; Viral; Viral Vector; Virus; adeno-associated viral vector; autism spectrum disorder; behavior influence; cell type; cost; cost effective; dopamine system; dopaminergic neuron; gene function; in vivo; interest; knockout gene; mature animal; motivated behavior; nervous system disorder; neural circuit; neuropsychiatric disorder; neurotransmitter release; novel; optogenetics; promoter; rapid technique; relating to nervous system; response; therapeutic target; trafficking; vector; voltage

Project Summary/Abstract Many types of neurons utilize complex and highly specific patterns of action potential firing to regulate neurotransmitter release and communicate with downstream cells. Action potential firing is primarily regulated through neurotransmitter input as well as through the expression of ion channel genes that determine the baseline firing properties of an individual neuron. While some of these ion channels are well studied, many remain unexplored or underexplored. Dopamine neurons in the ventral tegmental area (VTA) exhibit tightly controlled activity patterns, including bursts and pauses in activity, which encode information about environmental cues and rewards and influence learning and motivation. It is believed that disruptions in these firing patterns contribute to a variety of behavioral perturbations associated with mental illness. Thus, VTA dopamine neurons provide an excellent model system for investigating the function of understudied ion channels within their native neuronal environment. A recent study identified the entire complement of ion channels expressed in dopamine neurons in mice; I have selected for study three of these channels that also appear on the Illuminating the Druggable Genome (IDG) list of understudied proteins: Cacna2d3, encoding the 2-3 calcium channel auxiliary subunit, Kcna6, encoding the Shaker potassium channel KV1.6, and Kcnab2, encoding the KV2 potassium channel auxiliary subunit. I will utilize a novel, single vector adeno associated viral system that takes advantage of CRISPR/Cas9 gene editing technology to rapidly induce gene mutation in a cell-type specific (Cre-dependent) manner in neurons of adult mice. I will then use slice electrophysiology and fast-scan cyclic voltammetry in combination with optogenetics to characterize the effects of individual ion channel gene knockout and determine how these understudied ion channels regulate dopamine neuron physiology and dopamine release dynamics. Completion of this research will establish a simple, single virus technique for rapidly and specifically inducing gene knockout, which will be widely applicable to investigations of understudied proteins. Additionally, by identifying novel regulators of dopamine firing patterns we will both increase our understanding of the underlying physiology driving these critical neurons and identify new potential targets for precision therapeutics.

项目概要/摘要 许多类型的神经元利用复杂且高度特异性的动作电位放电模式来调节 神经递质的释放和与下游细胞的交流主要受到调节。 通过神经递质输入以及通过离子通道基因的表达来决定 虽然其中一些离子通道已得到充分研究，但许多离子通道的基线放电特性。 腹侧被盖区（VTA）的多巴胺神经元仍未被探索或探索不足。 受控的活动模式，包括活动的爆发和暂停，这些模式对有关的信息进行编码 据信，环境线索和奖励会影响学习和动机。 放电模式会导致与精神疾病相关的各种行为干扰。 多巴胺神经元为研究待研究离子的功能提供了一个出色的模型系统 最近的一项研究确定了其天然神经环境中的完整离子通道。 小鼠多巴胺神经元中表达的通道；我选择了其中三个通道进行研究 出现在阐明药物基因组 (IDG) 待研究蛋白质列表中：Cacna2d3，编码 2-3 钙通道辅助亚基 Kcna6，编码 Shaker 钾通道 KV1.6 和 Kcnab2， 编码 KV2 钾通道辅助亚基 I 将利用一种新颖的、单向量腺相关的。 利用CRISPR/Cas9基因编辑技术快速诱导基因突变的病毒系统 然后我将使用切片电生理学来研究成年小鼠神经元中细胞类型特异性（Cre 依赖性）的方式。 快速扫描循环伏安法与光遗传学相结合来表征单个离子的影响 通道基因敲除并确定这些正在研究的离子通道如何调节多巴胺神经元 生理学和多巴胺释放动力学的研究完成后将建立一种简单的单一病毒。 快速、特异性诱导基因敲除的技术，将广泛应用于研究 此外，通过识别多巴胺放电模式的新调节因子，我们将能够 增加我们对驱动这些关键神经元的潜在生理学的理解，并识别新的 精准治疗的潜在目标。

项目成果

The potassium channel auxiliary subunit Kvβ2 (Kcnab2) regulates Kv1 channels and dopamine neuron firing.

钾通道辅助亚基 Kvβ2 (Kcnab2) 调节 Kv1 通道和多巴胺神经元放电。

• DOI: 10.1152/jn.00194.2022
• 发表时间: 2022
• 期刊: Journal of neurophysiology
• 影响因子: 2.5
• 作者: Yee,JoshuaX;Rastani,Ariana;Soden,MartaE
• 通讯作者: Soden,MartaE

Temporal scaling of dopamine neuron firing and dopamine release by distinct ion channels shape behavior.

• DOI: 10.1126/sciadv.adg8869
• 发表时间: 2023-08-11
• 期刊: SCIENCE ADVANCES
• 影响因子: 13.6
• 作者: Juarez, Barbara;Kong, Mi-Seon;Jo, Yong S.;Elum, Jordan E.;Yee, Joshua X.;Ng-Evans, Scott;Cline, Marcella;Hunker, Avery C.;Quinlan, Meagan A.;Baird, Madison A.;Elerding, Abigail J.;Johnson, Mia;Ban, Derek;Mendez, Adriana;Goodwin, Nastacia L.;Soden, Marta E.;Zweifel, Larry S.
• 通讯作者: Zweifel, Larry S.