The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of PKA is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorometry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We found that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that nonphysiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto-unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.

中文翻译：

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Ca2+ 通道 CatSper 不会被 cAMP/PKA 信号激活，而是直接受用于探测 cAMP 和 PKA 作用的化学物质的影响。

精子特异性 Ca2+ 通道 CatSper（精子的阳离子通道）控制 Ca2+ 流入鞭毛，从而控制精子的游动行为。人类 CatSper 的一个标志是它通过膜电压、细胞内 pH 值和输卵管激素的多模式激活。然而，CatSper 是否也被涉及 cAMP 增加和随后激活 PKA 的信号通路激活是一个有争议的问题。为了阐明这个问题，我们使用动态离子敏感荧光测定法、膜片钳记录和光化学来研究来自健康供体和缺乏功能性 CatSper 通道的患者的人类精子的跨膜 Ca2+ 通量和膜电流。我们发现人类 CatSper 既不会被细胞内 cAMP 直接激活，也不会被 cAMP/PKA 信号通路间接激活。反而，我们展示了用于模拟细胞内 cAMP 作用的非生理浓度的 cAMP 和可渗透膜的 cAMP 类似物，通过迄今为止未知的细胞外结合位点从外部激活人类 CatSper。最后，我们证明了普通 PKA 抑制剂对人类 CatSper 的影响主要取决于（如果不是唯一的话）对 CatSper 本身的脱靶药物作用，而不是对 PKA 的抑制。我们得出结论，CatSper 的细胞内 cAMP/PKA 激活的概念主要基于用于干扰 cAMP 信号传导的化学探针的非特异性作用。总之，我们的研究结果解决了几个有争议的问题，并揭示了一个控制 CatSper 活性的新型配体结合位点，这对未来精子中 cAMP 和 Ca2+ 信号传导的研究具有重要意义。