Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
PLoS One. 2018 Dec 17;13(12):e0201322. doi: 10.1371/journal.pone.0201322. eCollection 2018.
Group IVa cytosolic phospholipase A2 (cPLA2α) mediates GPCR-stimulated arachidonic acid (AA) release from phosphatidylinositol 4,5-bisphosphate (PIP2) located in plasma membranes. We previously found in superior cervical ganglion (SCG) neurons that PLA2 activity is required for voltage-independent N-type Ca2+ (N-) current inhibition by M1 muscarinic receptors (M1Rs). These findings are at odds with an alternative model, previously observed for M-current inhibition, where PIP2 dissociation from channels and subsequent metabolism by phospholipase C suffices for current inhibition. To resolve cPLA2α's importance, we have investigated its role in mediating voltage-independent N-current inhibition (~40%) that follows application of the muscarinic agonist oxotremorine-M (Oxo-M). Preincubation with different cPLA2α antagonists or dialyzing cPLA2α antibodies into cells minimized N-current inhibition by Oxo-M, whereas antibodies to Ca2+-independent PLA2 had no effect. Taking a genetic approach, we found that SCG neurons from cPLA2α-/- mice exhibited little N-current inhibition by Oxo-M, confirming a role for cPLA2α. In contrast, cPLA2α antibodies or the absence of cPLA2α had no effect on voltage-dependent N-current inhibition by M2/M4Rs or on M-current inhibition by M1Rs. These findings document divergent M1R signaling mediating M-current and voltage-independent N-current inhibition. Moreover, these differences suggest that cPLA2α acts locally to metabolize PIP2 intimately associated with N- but not M-channels. To determine cPLA2α's functional importance more globally, we examined action potential firing of cPLA2α+/+ and cPLA2α-/- SCG neurons, and found decreased latency to first firing and interspike interval resulting in a doubling of firing frequency in cPLA2α-/- neurons. These unanticipated findings identify cPLA2α as a tonic regulator of neuronal membrane excitability.
IVa 组细胞溶质磷脂酶 A2（cPLA2α）介导 G 蛋白偶联受体刺激的花生四烯酸（AA）从位于质膜的磷脂酰肌醇 4,5-二磷酸（PIP2）释放。我们之前在颈上神经节（SCG）神经元中发现，PLA2 活性是 M1 毒蕈碱受体（M1R）对电压无关型 N 型钙（N-）电流抑制所必需的。这些发现与之前观察到的 M 电流抑制的替代模型不一致，在该模型中，通道与 PIP2 的解离以及随后的磷脂酶 C 代谢足以抑制电流。为了解决 cPLA2α 的重要性，我们研究了它在介导电压无关型 N 电流抑制（约 40%）中的作用，该抑制紧随毒蕈碱激动剂 Oxotremorine-M（Oxo-M）的应用而发生。用不同的 cPLA2α 拮抗剂预孵育或向细胞内透析 cPLA2α 抗体可使 Oxo-M 对 N 电流的抑制作用最小化，而对钙非依赖性 PLA2 的抗体则没有作用。通过遗传方法，我们发现 cPLA2α-/- 小鼠的 SCG 神经元对 Oxo-M 的 N 电流抑制作用很小，这证实了 cPLA2α 的作用。相比之下，cPLA2α 抗体或缺乏 cPLA2α 对 M2/M4R 对电压依赖性 N 电流的抑制作用或 M1R 对 M 电流的抑制作用没有影响。这些发现证明了介导 M 电流和电压无关型 N 电流抑制的不同 M1R 信号。此外，这些差异表明 cPLA2α 局部作用于代谢与 N 而非 M 通道密切相关的 PIP2。为了更全面地确定 cPLA2α 的功能重要性，我们检查了 cPLA2α+/+ 和 cPLA2α-/-SCG 神经元的动作电位放电，并发现 cPLA2α-/-神经元的首次放电潜伏期和尖峰间隔时间缩短，导致放电频率增加一倍。这些意想不到的发现确定 cPLA2α 是神经元膜兴奋性的紧张调节剂。
