光遗传学工具用于操纵与环核苷酸门控通道功能偶联的环核苷酸。

Optogenetic tools for manipulation of cyclic nucleotides functionally coupled to cyclic nucleotide-gated channels.

机构信息

Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Germany.

Institute of Biophysical Chemistry, Goethe University, Frankfurt, Germany.

出版信息

Br J Pharmacol. 2022 Jun;179(11):2519-2537. doi: 10.1111/bph.15445. Epub 2021 May 6.

DOI:10.1111/bph.15445

PMID:33733470

Abstract

BACKGROUND AND PURPOSE

The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers regulating numerous biological processes. Malfunctional cNMP signalling is linked to diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in Caenorhabditis elegans is restricted to soluble adenylyl cyclases, the membrane-bound Blastocladiella emersonii CyclOp and hyperpolarizing rhodopsins; yet missing are membrane-bound photoactivatable adenylyl cyclases and hyperpolarizers based on K currents.

EXPERIMENTAL APPROACH

For the characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with cyclic nucleotide-gated channels in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to depolarize or hyperpolarize cells, we performed behavioural analyses, measured cNMP content in vitro, and compared in vivo expression levels.

KEY RESULTS

We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine-control of cGMP levels. We established photoactivatable membrane-bound adenylyl cyclases, based on mutated versions ("A-2x") of Blastocladiella and Catenaria ("Be," "Ca") CyclOp, as N-terminal YFP fusions, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two-component optogenetics, we introduced the cAMP-gated K -channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A-2x), or YFP-BeCyclOp(A-2x). As an alternative, we implemented the B. emersonii cGMP-gated K -channel BeCNG1 together with BeCyclOp.

CONCLUSION AND IMPLICATIONS

We established a comprehensive suite of optogenetic tools for cNMP manipulation, applicable in many cell types, including sensory neurons, and for potent hyperpolarization.

LINKED ARTICLES

This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

摘要

背景与目的

环核苷酸 cAMP 和 cGMP 是调节众多生物过程的普遍第二信使。功能失调的 cNMP 信号与疾病有关，因此是药物研究的重要靶点。秀丽隐杆线虫现有的光遗传学工具仅限于可溶性腺苷酸环化酶、膜结合 Blastocladiella emersonii CyclOp 和超极化视紫红质；然而，缺少的是基于 K 电流的膜结合光激活腺苷酸环化酶和超极化剂。

实验方法

为了表征光激活的核苷酸环化酶，我们单独或与肌细胞和胆碱能运动神经元中的环核苷酸门控通道一起表达这些蛋白。为了研究光遗传学 cNMP 产生的程度以及这些系统去极化或超极化细胞的能力，我们进行了行为分析，测量了体外 cNMP 含量，并比较了体内表达水平。

主要结果

我们将 Catenaria CyclOp 作为 cGMP 产生的新工具，实现了 cGMP 水平的精细控制。我们建立了基于 Blastocladiella 和 Catenaria（“Be”、“Ca”）CyclOp 的突变体（“A-2x”）的光激活膜结合腺苷酸环化酶，作为 N 端 YFP 融合物，与可溶性 bPAC 相比，尽管整体 cAMP 产量较低，但能更有效和特异地进行 cAMP 信号传递。对于通过双组分光遗传学使可兴奋细胞超极化，我们引入了来自 Spirochaeta thermophila 的 cAMP 门控 K -通道 SthK，并将其与 bPAC、BeCyclOp（A-2x）或 YFP-BeCyclOp（A-2x）结合。作为替代方案，我们实现了 B. emersonii 的 cGMP 门控 K -通道 BeCNG1 与 BeCyclOp 结合。