Tweety proteins: their roles in pericytes and macrophages

神经元或肌肉肌细胞中的离子通道、转运蛋白、交换蛋白和泵调节 动作电位和收缩活动。在内分泌细胞中，它们调节激素分泌。离子通道具有 甚至在非兴奋性细胞中也起着关键作用，例如，通过设置膜电位和调节 Ca2+进入细胞。有数百个渠道，大多数都有相对较好的特点。然而，有一些人， 仍然未得到充分研究，这是RFA-RM-22 - 024的重点。我们实验室的一个重点领域是研究 周细胞是毛细血管腔外表面的特化细胞。周细胞有多个 功能，包括形成新血管和调节血液流动。周细胞也是 先天免疫系统，并通过分泌趋化因子直接与几种类型的免疫细胞相互作用， 细胞因子，包括IFN-γ、TNF-α、IL-1 β和IL-6。我们已经开发了一种小鼠模型，其中mCherry是 在周细胞中明确表达，这允许分离周细胞并富集至高纯度。我们有 进行了全局RNA-seq，并专注于> 650个离子通道，交换剂和 泵在脑周细胞中表达。几个Cl-和K+通道的转录本。值得注意的是， TTYH1、TTYH2和TTYH3是脑周细胞中最高表达的通道。 这一发现得到了公共数据库的证实，表明Tthy2特异性表达于乳腺癌的周细胞。 成年小鼠肺血管和血管周围细胞。Tthy2最初被表征为肿胀依赖性 体积调节阴离子通道，但后来的冷冻电镜研究无法确定结构特征， 这与阴离子传导孔的已知特征一致。我们假设Tthy2可能是一个 它可能是体积调节阴离子通道的组分或调节剂，或者它可能具有非通道功能。在目标1中， 我们将研究Tweety蛋白是否作为微血管周细胞中阴离子通道的组分。我们 将记录来自原代人脑血管周细胞的容量调节阴离子电流（VRAC），并比较 具有或不具有TTYH2的CRISPR敲低的数据。实验也被设计来研究其他类型的 水流在目标2中，我们将跟进提示TTYH2作为免疫系统参与的初步研究结果。 哨兵。具体来说，我们发现TTYH2可能是cGAS-STING通路的负调节因子， 控制IFN-β和IL-6响应外源（例如，病毒）DNA。我们将测试 通过检查刺激后IRF3磷酸化和IFN-β I型IFN和IL-6的产生来观察TTYH2的功能 通过用cGAMP或STING激动剂DMXAA处理细胞来抑制cGAS-STING途径。这种多PI R03 William Coetzee博士和Stefan Feske博士的建议汇集了他们独特的专业知识，以更好地了解 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- 针对免疫性疾病的离子通道的获批药物，以及拟议研究的完成 让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.