Neurophysiology Research Center, Shahid Beheshti University of Medical Sciences, Evin, Tehran 19857, Iran; Department of Physiology, Shahid Beheshti University of Medical Sciences, Evin, Tehran 19857, Iran.
Neurobiology Research Center, Shahid Beheshti University of Medical Sciences, Evin, Tehran 19857, Iran.
Exp Neurol. 2015 Jul;269:8-16. doi: 10.1016/j.expneurol.2014.12.024. Epub 2015 Mar 28.
Mitochondrial dysfunction is a hallmark of amyloid-beta (Aβ)-induced neuronal toxicity in Alzheimer's disease (AD). However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain. Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was undertaken to investigate whether the gating behavior of the mitochondrial ATP- and ChTx-insensitive-IbTx-sensitive Ca(2+)-activated potassium channel (mitoBKCa) is altered in a rat model of Aβ neurotoxicity. Aβ1-42 (4 μg/μl) was intracerebroventricularly injected in male Wistar rats (220-250 g). Brain Aβ accumulation was confirmed two weeks later on the basis of an immunohistochemistry staining assay, and physiological impacts measured in passive avoidance task cognitive performance experiments. Brain mitochondrial inner membranes were then extracted and membrane vesicles prepared for channel incorporation into bilayer lipid. Purity of the cell fraction was confirmed by Western blot using specific markers of mitochondria, plasma membrane, endoplasmic reticulum, and Golgi. Our results first provide evidence for differences in mitoBKCa ion permeation properties with channels coming from Aβ vesicle preparations characterized by an inward rectifying I-V curve, in contrast to control mitoBKCa channels which showed a linear I-V relationship under the same ionic conditions (200 mM cis/50mM trans). More importantly the open probability of channels from Aβ vesicles appeared 1.5 to 2.5 smaller compared to controls, the most significant decrease being observed at depolarizing potentials (30 mV to 50 mV). Because BKCa-β4 subunit has been documented to shift the BKCa channel voltage dependence curve, a Western blot analysis was undertaken where expression of mitoBKCa α and β4 subunits was estimated using anti-α and β4 subunit antibodies. Our results indicated a significant increase in mitoBKCa-β4 subunit expression coupled to a decrease in the expression of α subunit. Our results thus demonstrate a modification in the mitoBKCa channel gating properties in membrane preparations coming from a rat model of Aβ neurotoxicity, an effect potentially linked to a change in mitoBKCa-β4 and -α subunits expression or increased ROS production due to an enhanced Aβ mitochondrial accumulation. Our results may provide new insights into the cellular mechanisms underlying mitochondrial dysfunctions in Aβ neurotoxicity.
线粒体功能障碍是阿尔茨海默病（AD）中β淀粉样蛋白（Aβ）诱导神经元毒性的标志。然而，Aβ如何影响线粒体功能的潜在机制仍不确定。由于线粒体钾通道参与了几种线粒体功能，包括细胞保护、凋亡和钙稳态，因此进行了一项研究，以调查在 Aβ神经毒性的大鼠模型中，线粒体 ATP 和 ChTx 不敏感-IbTx 敏感 Ca（2+）激活的钾通道（mitoBKCa）的门控行为是否发生改变。Aβ1-42（4μg/μl）经侧脑室注射雄性 Wistar 大鼠（220-250g）。两周后，基于免疫组织化学染色测定证实大脑中 Aβ 的积累，然后在被动回避任务认知性能实验中测量生理影响。然后从大脑线粒体内膜中提取膜囊泡，并准备用于通道掺入双层脂质。使用线粒体、质膜、内质网和高尔基体的特异性标记物通过 Western blot 确认细胞级分的纯度。我们的结果首先提供了证据，证明来自 Aβ 囊泡制剂的 mitoBKCa 离子渗透特性存在差异，这些囊泡制剂的通道表现为内向整流的 I-V 曲线，而对照 mitoBKCa 通道在相同的离子条件下（200mM cis/50mM trans）表现出线性的 I-V 关系。更重要的是，与对照相比，来自 Aβ 囊泡的通道的开放概率降低了 1.5 到 2.5 倍，在去极化电位（30 mV 至 50 mV）下观察到最大的降低。由于已经记录到 BKCa-β4 亚基改变 BKCa 通道的电压依赖性曲线，因此进行了 Western blot 分析，使用抗-α 和 β4 亚基抗体估计 mitoBKCa α 和 β4 亚基的表达。我们的结果表明，mitoBKCa-β4 亚基的表达显著增加，而 α 亚基的表达减少。因此，我们的结果表明，在来自 Aβ 神经毒性大鼠模型的膜制剂中，mitoBKCa 通道的门控特性发生了改变，这种效应可能与 mitoBKCa-β4 和-α 亚基表达的变化或由于 Aβ 线粒体积累增加而导致的 ROS 产生增加有关。我们的结果可能为 Aβ 神经毒性中线粒体功能障碍的细胞机制提供新的见解。
