Carmel L Howe, David Icka-Araki, Alexander E G Viray, Sarahi Garza, James A Frank
{"title":"利用系留光药理学对体外 TRPV1 通道进行光学控制。","authors":"Carmel L Howe, David Icka-Araki, Alexander E G Viray, Sarahi Garza, James A Frank","doi":"10.1021/acschembio.4c00052","DOIUrl":null,"url":null,"abstract":"<p><p>Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel that is important for nociception and inflammatory pain and is activated by a variety of nociceptive stimuli─including lipids such as capsaicin (CAP) and endocannabinoids. TRPV1's role in physiological systems is often studied by activating it with externally perfused ligands; however, this approach is plagued by poor spatiotemporal resolution. Lipid agonists are insoluble in physiological buffers and can permeate membranes to accumulate nonselectively inside cells, where they can have off-target effects. To increase the spatiotemporal precision with which we can activate lipids on cells and tissues, we previously developed optically cleavable targeted (OCT) ligands, which use protein tags (SNAP-tags) to localize a photocaged ligand on a target cellular membrane. After enrichment, the active ligand is released on a flash of light to activate nearby receptors. In our previous work, we developed an OCT-ligand to control a cannabinoid-sensitive GPCR. Here, we expand the scope of OCT-ligand technology to target TRPV1 ion channels. We synthesize a probe, OCT-CAP, that tethers to membrane-bound SNAP-tags and releases a TRPV1 agonist when triggered by UV-A irradiation. Using Ca<sup>2+</sup> imaging and electrophysiology in HEK293T cells expressing TRPV1, we demonstrate that OCT-CAP uncaging activates TRPV1 with superior spatiotemporal precision when compared to standard diffusible ligands or photocages. This study is the first example of an OCT-ligand designed to manipulate an ion-channel target. We anticipate that these tools will find many applications in controlling lipid signaling pathways in various cells and tissues.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5000,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Optical Control of TRPV1 Channels <i>In Vitro</i> with Tethered Photopharmacology.\",\"authors\":\"Carmel L Howe, David Icka-Araki, Alexander E G Viray, Sarahi Garza, James A Frank\",\"doi\":\"10.1021/acschembio.4c00052\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel that is important for nociception and inflammatory pain and is activated by a variety of nociceptive stimuli─including lipids such as capsaicin (CAP) and endocannabinoids. TRPV1's role in physiological systems is often studied by activating it with externally perfused ligands; however, this approach is plagued by poor spatiotemporal resolution. Lipid agonists are insoluble in physiological buffers and can permeate membranes to accumulate nonselectively inside cells, where they can have off-target effects. To increase the spatiotemporal precision with which we can activate lipids on cells and tissues, we previously developed optically cleavable targeted (OCT) ligands, which use protein tags (SNAP-tags) to localize a photocaged ligand on a target cellular membrane. After enrichment, the active ligand is released on a flash of light to activate nearby receptors. In our previous work, we developed an OCT-ligand to control a cannabinoid-sensitive GPCR. Here, we expand the scope of OCT-ligand technology to target TRPV1 ion channels. We synthesize a probe, OCT-CAP, that tethers to membrane-bound SNAP-tags and releases a TRPV1 agonist when triggered by UV-A irradiation. Using Ca<sup>2+</sup> imaging and electrophysiology in HEK293T cells expressing TRPV1, we demonstrate that OCT-CAP uncaging activates TRPV1 with superior spatiotemporal precision when compared to standard diffusible ligands or photocages. This study is the first example of an OCT-ligand designed to manipulate an ion-channel target. We anticipate that these tools will find many applications in controlling lipid signaling pathways in various cells and tissues.</p>\",\"PeriodicalId\":11,\"journal\":{\"name\":\"ACS Chemical Biology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.5000,\"publicationDate\":\"2024-06-21\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ACS Chemical Biology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1021/acschembio.4c00052\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Chemical Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1021/acschembio.4c00052","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
瞬时受体电位类香草素 1(TRPV1)是一种非选择性阳离子通道,对痛觉和炎症性疼痛非常重要,可被多种痛觉刺激物激活--包括脂质,如辣椒素(CAP)和内源性大麻素。研究 TRPV1 在生理系统中的作用时,通常使用外部灌注的配体激活 TRPV1;然而,这种方法存在时空分辨率低的问题。脂质激动剂不溶于生理缓冲液,可穿透细胞膜在细胞内非选择性聚集,产生脱靶效应。为了提高激活细胞和组织上脂质的时空精确度,我们之前开发了光学可裂解靶向(OCT)配体,这种配体使用蛋白质标签(SNAP-标签)将光笼配体定位在目标细胞膜上。富集后,活性配体在闪光时释放出来,激活附近的受体。在我们之前的工作中,我们开发了一种 OCT 配体来控制大麻素敏感的 GPCR。在这里,我们将 OCT 配体技术的范围扩大到针对 TRPV1 离子通道。我们合成了一种探针 OCT-CAP,它能与膜结合的 SNAP 标签相连,并在 UV-A 照射触发时释放 TRPV1 激动剂。通过在表达 TRPV1 的 HEK293T 细胞中进行 Ca2+ 成像和电生理学研究,我们证明了与标准扩散配体或光电笼相比,OCT-CAP 能以更高的时空精度激活 TRPV1。这项研究是首个利用 OCT 配体操纵离子通道靶点的实例。我们预计,这些工具将在控制各种细胞和组织的脂质信号通路中得到广泛应用。
Optical Control of TRPV1 Channels In Vitro with Tethered Photopharmacology.
Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel that is important for nociception and inflammatory pain and is activated by a variety of nociceptive stimuli─including lipids such as capsaicin (CAP) and endocannabinoids. TRPV1's role in physiological systems is often studied by activating it with externally perfused ligands; however, this approach is plagued by poor spatiotemporal resolution. Lipid agonists are insoluble in physiological buffers and can permeate membranes to accumulate nonselectively inside cells, where they can have off-target effects. To increase the spatiotemporal precision with which we can activate lipids on cells and tissues, we previously developed optically cleavable targeted (OCT) ligands, which use protein tags (SNAP-tags) to localize a photocaged ligand on a target cellular membrane. After enrichment, the active ligand is released on a flash of light to activate nearby receptors. In our previous work, we developed an OCT-ligand to control a cannabinoid-sensitive GPCR. Here, we expand the scope of OCT-ligand technology to target TRPV1 ion channels. We synthesize a probe, OCT-CAP, that tethers to membrane-bound SNAP-tags and releases a TRPV1 agonist when triggered by UV-A irradiation. Using Ca2+ imaging and electrophysiology in HEK293T cells expressing TRPV1, we demonstrate that OCT-CAP uncaging activates TRPV1 with superior spatiotemporal precision when compared to standard diffusible ligands or photocages. This study is the first example of an OCT-ligand designed to manipulate an ion-channel target. We anticipate that these tools will find many applications in controlling lipid signaling pathways in various cells and tissues.
期刊介绍:
ACS Chemical Biology provides an international forum for the rapid communication of research that broadly embraces the interface between chemistry and biology.
The journal also serves as a forum to facilitate the communication between biologists and chemists that will translate into new research opportunities and discoveries. Results will be published in which molecular reasoning has been used to probe questions through in vitro investigations, cell biological methods, or organismic studies.
We welcome mechanistic studies on proteins, nucleic acids, sugars, lipids, and nonbiological polymers. The journal serves a large scientific community, exploring cellular function from both chemical and biological perspectives. It is understood that submitted work is based upon original results and has not been published previously.