Kirchner Matthew K, Armstrong William E, Guan Dongxu, Ueta Yoichi, Foehring Robert C
Department of Anatomy and Physiology, University of Tennessee Health Science Center, Memphis, Tennessee.
Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
Physiol Rep. 2019 Aug;7(16):e14198. doi: 10.14814/phy2.14198.
Magnocellular neurosecretory cells (MNCs) occupying the supraoptic nucleus (SON) contain voltage-gated Ca channels that provide Ca for triggering vesicle release, initiating signaling pathways, and activating channels, such as the potassium channels underlying the afterhyperpolarization (AHP). Phosphotidylinositol 4,5-bisphosphate (PIP ) is a phospholipid membrane component that has been previously shown to modulate Ca channels, including in the SON in our previous work. In this study, we further investigated the ways in which PIP modulates these channels, and for the first time show how PIP modulates Ca channel currents in native membranes. Using whole cell patch clamp of genetically labeled dissociated neurons, we demonstrate that PIP depletion via wortmannin (0.5 μmol/L) inhibits Ca channel currents in OT but not VP neurons. Additionally, it hyperpolarizes voltage-dependent activation of the channels by ~5 mV while leaving the slope of activation unchanged, properties unaffected in VP neurons. We also identified key differences in baseline currents between the cell types, wherein VP whole cell Ca currents display more inactivation and shorter deactivation time constants. Wortmannin accelerates inactivation of Ca channels in OT neurons, which we show to be mostly an effect on N-type Ca channels. Finally, we demonstrate that wortmannin prevents prepulse-induced facilitation of peak Ca channel currents. We conclude that PIP is a modulator that enhances current through N-type channels. This has implications for the afterhyperpolarization (AHP) of OT neurons, as previous work from our laboratory demonstrated the AHP is inhibited by wortmannin, and that its primary activation is from intracellular Ca contributed by N-type channels.
占据视上核（SON）的大细胞神经分泌细胞（MNCs）含有电压门控钙通道，这些通道为触发囊泡释放、启动信号通路以及激活通道（如超极化后电位（AHP）背后的钾通道）提供钙离子。磷脂酰肌醇4,5-二磷酸（PIP₂）是一种磷脂膜成分，先前的研究表明它可以调节钙通道，包括在我们之前的工作中对视上核中的钙通道进行调节。在本研究中，我们进一步研究了PIP₂调节这些通道的方式，并首次展示了PIP₂如何调节天然膜中的钙通道电流。通过对基因标记的解离神经元进行全细胞膜片钳记录，我们证明，通过渥曼青霉素（0.5 μmol/L）耗尽PIP₂会抑制催产素（OT）神经元而非加压素（VP）神经元的钙通道电流。此外，它使通道的电压依赖性激活超极化约5 mV，同时激活斜率不变，VP神经元的这些特性不受影响。我们还确定了不同细胞类型在基线电流方面的关键差异，其中VP全细胞钙电流表现出更多的失活和更短的失活时间常数。渥曼青霉素加速OT神经元中钙通道的失活，我们发现这主要是对N型钙通道的影响。最后，我们证明渥曼青霉素可阻止预脉冲诱导的钙通道电流峰值增强。我们得出结论，PIP₂是一种增强通过N型通道电流的调节剂。这对视上核神经元的超极化后电位（AHP）具有重要意义，因为我们实验室之前的研究表明，渥曼青霉素可抑制AHP，并且其主要激活来自N型通道贡献的细胞内钙离子。
