Tweety proteins: their roles in pericytes and macrophages

神经元或肌肉细胞中的离子通道、转运体、交换器和泵调节 动作电位和收缩活动。在内分泌细胞中，它们调节激素的分泌。离子通道具有 甚至在不可兴奋的细胞中也起着关键作用，例如，通过设置膜电位和调节内流 CA2进入细胞。有数百个频道，大多数都有相对较好的特点。然而，一个数字， 仍未得到充分研究，这是RFA-RM-22-024的重点。我们实验室的一个重点领域是调查 周细胞，毛细血管管腔表面的特化细胞。周细胞有多个 功能，包括形成新的血管和调节血液流动。周细胞也是 与生俱来的免疫系统，通过分泌趋化因子和 细胞因子，包括干扰素-γ、肿瘤坏死因子-α、白介素1β和白介素6。我们已经开发了一个小鼠模型，在这个模型中，mCherry是 在周细胞中明确表达，这允许分离周细胞并浓缩到高纯度。我们有 执行全球RNA-SEQ，并专注于&gt；650离子通道、交换器和 泵在脑周细胞中表达。存在几种氯离子和钾离子通道的转录本。注意了，各位成员 Twety家族成员(TTYH1、TTYH2和TTYH3)是脑周细胞最高表达通道之一。 这一发现得到了公共数据库的证实，表明Thy2特异地表达于 成年小鼠肺血管及血管周围细胞。Thy2最初的特征是肿胀依赖。 容量调节的阴离子通道，但后来的冷冻-EM研究无法确定 与已知的阴离子导电孔的特征一致。我们假设Thy2可能是一个 体积调节阴离子通道的组件或调节器，或者它可以具有非通道功能。在目标1中， 我们将研究Twety蛋白是否作为微血管周细胞阴离子通道的组成部分。我们 将记录原代人脑血管周细胞的容量调节阴离子电流(Vrac)并比较 有或没有CRISPR击倒TTYH2的数据。实验也被设计用来研究其他类型的 洋流。在目标2中，我们将跟进初步研究结果，表明TTYH2作为一种免疫参与 哨兵。具体地说，我们发现TTYH2可能是cGAS-STING途径的负调控因子，这可能是cGAS-STING途径的负调控因子 控制干扰素-β和IL-6的产生，以响应外来的(如病毒)DNA。我们将进行生理测试 TTYH2通过检测刺激后I型干扰素和IL-6产生及IRF3的磷酸化来发挥作用 CGAMP或刺激剂DMXAA处理细胞的cGAS-刺痛途径。这款多路PI R03 William Coetzee博士和Stefan Feske博士的提案将他们独特的专业知识结合在一起，以更好地理解 通道，特别是TTYH2，在血管功能和先天免疫中的作用。目前没有FDA- 针对免疫紊乱的离子通道的批准药物，并完成拟议的研究 使我们朝着这个错失的治疗机会迈出了重要的一步。

Ion channels, transporters, exchangers, and pumps in neurons or muscle myocytes regulate the formation of action potentials and contractile activity. In endocrine cells, they regulate hormone secretion. Ion channels have key roles even in non-excitable cells, for example, by setting the membrane potential and regulating the influx of Ca2+ into cells. There are hundreds of channels, and most are relatively well characterized. A number, however, remains understudied, which is the focus of RFA-RM-22-024. One focus area of our lab is to investigate the roles of pericytes, specialized cells on the abluminal surface of capillary blood vessels. Pericytes have multiple functions, including forming new blood vessels and regulating blood flow. Pericytes are also sentinels of the innate immune system and directly interact with several types of immune cells by secreting chemokines and cytokines, including IFN-γ, TNF-α, IL-1β, and IL-6. We have developed a mouse model in which mCherry is explicitly expressed in pericytes, which allows isolation of pericytes and enrichment to high purity. We have performed global RNA-seq and focused on the transcriptional profiles of >650 ion channels, exchangers, and pumps expressed in brain pericytes. Transcripts of several Cl- and K+ channels were present. Of note, members of the tweety family (TTYH1, TTYH2, and TTYH3) were amongst the top expressing channels in brain pericytes. This finding was corroborated by public databases, showing that Tthy2 is specifically expressed in pericytes of adult mouse lung vascular and perivascular cells. Tthy2 was initially characterized as swelling-dependent volume-regulated anion channels, but later cryo-EM studies could not identify structural features that are consistent with known characteristics of an anion conduction pore. We hypothesize that Tthy2 might be a component or regulator of a volume-regulated anion channel or that it may have non-channel functions. In Aim 1, we will investigate whether tweety proteins act as components of anion channels in microvascular pericytes. We will record volume-regulated anion currents (VRAC) from primary human brain vascular pericytes and compare data with or without CRISPR knockdown of TTYH2. Experiments are also designed to investigate other types of currents. In Aim 2, we will follow up on preliminary findings suggesting that TTYH2 participates as an immune sentinel. Specifically, we found that TTYH2 may be a negative regulator of the cGAS-STING pathway, which controls the production of IFN-β and IL-6 in response to foreign (e.g., viral) DNA. We will test the physiological function of TTYH2 by examining IRF3 phosphorylation and IFN-β type I IFN and IL-6 production after stimulating the cGAS-STING pathway by treating cells with cGAMP or the STING agonist DMXAA. This multi-PI R03 proposal by Drs. William Coetzee and Stefan Feske bring together their unique expertise to better understand the roles of channels, particularly TTYH2, in vascular function and innate immunity. There are currently no FDA- approved drugs that target ion channels for immunological disorders, and the completion of the proposed studies takes us an essential step in the direction of this missed therapeutic opportunity.