Tweety proteins: their roles in pericytes and macrophages

神经元或肌肉细胞中的离子通道、转运蛋白、交换器和泵调节 动作电位和收缩活动。在内分泌细胞中，它们调节激素分泌。离子通道有 即使在非兴奋性细胞中也发挥着关键作用，例如，通过设置膜电位和调节细胞流入 Ca2+进入细胞。有数百个通道，并且大多数都具有相对较好的特征。然而，有数字表明， 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研究角色 周细胞，毛细血管管腔表面的特殊细胞。周细胞有多个 功能，包括形成新血管和调节血流。周细胞也是细胞的哨兵 先天免疫系统并通过分泌趋化因子和直接与多种类型的免疫细胞相互作用 细胞因子，包括 IFN-γ、TNF-α、IL-1β 和 IL-6。我们开发了一种小鼠模型，其中 mCherry 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 测序，重点关注 > 650 个离子通道、交换器和 泵在脑周细胞中表达。存在多个 Cl- 和 K 通道的转录本。值得注意的是，会员 tweety 家族的成员（TTYH1、TTYH2 和 TTYH3）是大脑周细胞中表达最高的通道之一。 这一发现得到了公共数据库的证实，表明 Tthy2 在以下细胞的周细胞中特异性表达： 成年小鼠肺血管和血管周围细胞。 Tthy2 最初被定性为肿胀依赖性 体积调节的阴离子通道，但后来的冷冻电镜研究无法识别结构特征 与阴离子传导孔的已知特征一致。我们假设 Tthy2 可能是 体积调节阴离子通道的组件或调节器，或者它可能具有非通道功能。在目标 1 中， 我们将研究tweety蛋白是否充当微血管周细胞中阴离子通道的组成部分。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）并进行比较 有或没有 CRISPR 敲低 TTYH2 的数据。实验还旨在研究其他类型的 电流。在目标 2 中，我们将跟进表明 TTYH2 作为免疫参与的初步发现 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 的产生，以响应外来（例如病毒）DNA。我们将进行生理测试 通过检查 IRF3 磷酸化和刺激后 IFN-β I 型 IFN 和 IL-6 的产生来了解 TTYH2 的功能 通过用 cGAMP 或 STING 激动剂 DMXAA 处理细胞来调节 cGAS-STING 通路。此多PI R03 博士的提议。 William Coetzee 和 Stefan Feske 汇集了他们独特的专业知识，以更好地理解 通道（尤其是 TTYH2）在血管功能和先天免疫中的作用。目前尚无FDA- 批准的靶向离子通道治疗免疫性疾病的药物，以及拟议研究的完成 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.