Department of Neurophysiology, Institute of Physiology, Biocenter, Julius-Maximilians-University of Würzburg, Röntgenring 9, 97070, Würzburg, Germany.
Institute for Synaptic Physiology, Center for Molecular Neurobiology Hamburg, University Medical Center Hamburg-Eppendorf, 20251, Hamburg, Germany.
BMC Biol. 2021 Oct 18;19(1):227. doi: 10.1186/s12915-021-01151-9.
Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that transduces extracellular signals in virtually all eukaryotic cells. The soluble Beggiatoa photoactivatable adenylyl cyclase (bPAC) rapidly raises cAMP in blue light and has been used to study cAMP signaling pathways cell-autonomously. But low activity in the dark might raise resting cAMP in cells expressing bPAC, and most eukaryotic cyclases are membrane-targeted rather than soluble. Our aim was to engineer a plasma membrane-anchored PAC with no dark activity (i.e., no cAMP accumulation in the dark) that rapidly increases cAMP when illuminated.
Using a streamlined method based on expression in Xenopus oocytes, we compared natural PACs and confirmed bPAC as the best starting point for protein engineering efforts. We identified several modifications that reduce bPAC dark activity. Mutating a phenylalanine to tyrosine at residue 198 substantially decreased dark cyclase activity, which increased 7000-fold when illuminated. Whereas Drosophila larvae expressing bPAC in mechanosensory neurons show nocifensive-like behavior even in the dark, larvae expressing improved soluble (e.g., bPAC(R278A)) and membrane-anchored PACs exhibited nocifensive responses only when illuminated. The plasma membrane-anchored PAC (PACmn) had an undetectable dark activity which increased >4000-fold in the light. PACmn does not raise resting cAMP nor, when expressed in hippocampal neurons, affect cAMP-dependent kinase (PKA) activity in the dark, but rapidly and reversibly increases cAMP and PKA activity in the soma and dendrites upon illumination. The peak responses to brief (2 s) light flashes exceed the responses to forskolin-induced activation of endogenous cyclases and return to baseline within seconds (cAMP) or ~10 min (PKA).
PACmn is a valuable optogenetic tool for precise cell-autonomous and transient stimulation of cAMP signaling pathways in diverse cell types.
环磷酸腺苷（cAMP）是一种普遍存在的第二信使，几乎可以在所有真核细胞中转导细胞外信号。可溶性 Beggiatoa 光激活腺苷酸环化酶（bPAC）可在蓝光下快速升高 cAMP，并已被用于自主研究 cAMP 信号通路。但是，在黑暗中活性较低可能会导致表达 bPAC 的细胞中静息 cAMP 升高，而大多数真核环化酶都是膜靶向的而不是可溶性的。我们的目标是设计一种无暗活性的（即在黑暗中不积累 cAMP）的质膜锚定的 PAC，当被光照时能迅速升高 cAMP。
使用基于 Xenopus oocytes 表达的简化方法，我们比较了天然 PAC，并证实 bPAC 是蛋白工程的最佳起点。我们确定了几种可以降低 bPAC 暗活性的修饰。将残基 198 处的苯丙氨酸突变为酪氨酸，可显著降低暗环化酶活性，当被光照时，活性增加了 7000 倍。尽管在机械感觉神经元中表达 bPAC 的果蝇幼虫即使在黑暗中也会表现出类似伤害性的行为，但表达改良的可溶性（例如 bPAC（R278A））和质膜锚定的 PAC 的幼虫只有在光照时才会出现伤害性反应。质膜锚定的 PAC（PACmn）在黑暗中几乎没有暗活性，而在光照下活性增加了 4000 倍以上。PACmn 不会升高静息 cAMP，也不会在表达于海马神经元时影响黑暗中的 cAMP 依赖性激酶（PKA）活性，但在光照下，它能迅速且可逆地增加胞体和树突中的 cAMP 和 PKA 活性。对短暂（2 秒）光闪烁的峰值反应超过了对 forskolin 诱导的内源性环化酶激活的反应，并在几秒钟（cAMP）或 ~10 分钟（PKA）内恢复到基线。
PACmn 是一种有价值的光遗传学工具，可用于精确自主和瞬时刺激多种细胞类型中的 cAMP 信号通路。
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