Multisite phosphorylation by protein kinase A mediates modality-specific modulation of PIEZO2

Protein kinase A is a downstream effector of many inflammatory mediators that induce pain hypersensitivity by increasing the mechanosensitivity of nociceptive sensory afferent. Here we examine the molecular mechanism underlying protein kinase-Adependent modulation of the mechanically-activated ion channel PIEZO2, which confers mechanosensitivity to many nociceptors. Using phosphorylation site prediction algorithms, we identified multiple putative and highly conserved PKA phosphorylation sites located on intracellular intrinsically disordered regions of PIEZO2. Site-directed mutagenesis and patch-clamp recordings showed that substitution of one or multiple putative PKA sites within a single intracellular domains does not alter PKA-induced PIEZO2 sensitization, whereas mutation of a combination of nine putative sites located on four different intracellular regions completely abolishes PKA-dependent PIEZO2 modulation, suggesting that the effect requires multisite phosphorylation. By demonstrating that PIEZO1 is not modulated by PKA, our data also reveals a previously unrecognized functional difference between PIEZO1 and PIEZO2. Moreover, by demonstrating that PKA only modulates PIEZO2 currents evoked by focal mechanical indentation of the cell, but not currents evoked by pressure-induced membrane stretch, we provide evidence suggesting that PIEZO2 is a polymodal mechanosensor that engages different protein domains for detecting different types of mechanical stimuli.

蛋白激酶A（Protein Kinase A, PKA）是多种炎症介质的下游效应因子，此类炎症介质可通过增强伤害感受性感觉传入纤维的机械敏感性，诱导痛觉超敏。本研究探讨了蛋白激酶A依赖性调控机械激活离子通道PIEZO2的分子机制，该通道可赋予多种伤害感受器机械敏感性。研究人员通过磷酸化位点预测算法，在PIEZO2的细胞内固有无序区域中鉴定出多个推定且高度保守的PKA磷酸化位点。定点诱变与膜片钳记录实验结果显示，替换单个细胞内结构域内的一个或多个推定PKA位点，并不会改变PKA诱导的PIEZO2敏化效应；而对分布于四个不同细胞内区域的九个推定位点进行联合突变，则可完全阻断PKA依赖性的PIEZO2调控，提示该调控效应依赖于多位点磷酸化。本研究证实PIEZO1不受PKA调控，这一结果揭示了PIEZO1与PIEZO2之间此前未被认知的功能差异。此外，本研究发现PKA仅能调控细胞局部机械压痕诱导的PIEZO2电流，而非压力诱导的膜牵张激活的PIEZO2电流，这为PIEZO2作为多模式机械传感器——通过不同蛋白质结构域感知不同类型的机械刺激——提供了实验依据。