辅酶Q过量设置下线粒体通透性转变孔隙的开放概率评估
Summary
该方法利用线粒体通透性转变孔隙对低导质子泄漏的贡献，确定与野生型对照相比，心肌细胞线粒体辅酶Q含量增加的新生儿脆性X综合征小鼠孔隙开放的电压阈值。
Abstract
线粒体通透性转变孔（mPTP）是一种电压门控，非选择性，线粒体内膜（IMM）巨型通道，在健康和疾病中很重要。mPTP介导质子在低电导开口期间通过IMM泄漏，并被环孢菌素A（CsA）特异性抑制。辅酶Q（CoQ）是mPTP的调节因子，在脆性X综合征（FXS， Fmr1 敲除）的新生小鼠模型中，已经发现前脑和心脏线粒体中CoQ含量和mPTP开放概率的组织特异性差异。我们开发了一种技术来确定这种突变应变中mPTP开口的电压阈值，利用mpTPP作为质子泄漏通道的作用。
为此，在泄漏呼吸期间，使用极谱法和四苯基膦（TPP +）离子选择性电极在分离的线粒体中同时测量氧消耗量和膜电位（ΔΨ）。mPTP打开的阈值由CsA介导的对特定膜电位处质子泄漏的抑制的发作决定。使用这种方法，在辅酶Q过量的背景下精确定义了mpTPP的电压门控差异。这种新技术将允许未来的研究，以增强对mpTPP低电导开口的生理和病理调节的理解。
Introduction
mPTP介导渗透率转变（PT），由此IMM突然变得对小分子和溶质1，2具有渗透性。这种惊人的现象明显背离了IMM的特征不渗透性，后者是建立氧化磷酸化3所需的电化学梯度的基础。与其他线粒体转运机制不同，PT是一种高电导，非特异性和非选择性过程，允许一系列分子通过高达1.5 kDa4，5。mPTP是IMM内的电压门控通道，其开口会改变ΔΨ，ATP产生，钙稳态，活性氧（ROS）产生和细胞活力4。
在病理极端，mPTP的不受控制和长时间的高电导开放导致电化学梯度的塌陷，基质肿胀，基质吡啶核苷酸的消耗，外膜破裂，膜间蛋白（包括细胞色素c）的释放，最终导致细胞死亡4，6。这种病理性 mPTP 开放与心肌缺血再灌注损伤、心力衰竭、创伤性脑损伤、各种神经退行性疾病和糖尿病有关1，7。然而，低电导 mPTP 开孔本质上是生理性的，与高导通度开路相比，不会导致严重的去极化或线粒体肿胀4.
孔隙的低电导开口将渗透率限制在~300 Da，允许质子独立于ATP合成通过，并且是生理质子泄漏的潜在来源5。生理mPTP开放导致ΔΨ的受控下降，通过呼吸运输链增加电子通量，并导致超氧化物的短爆发或闪光，有助于ROS信号传导8。调节这种短暂的mPTP开放对于钙稳态和正常细胞发育和成熟很重要4，9，10，11。例如，发育中神经元的瞬时孔隙打开触发分化，而mPTP的闭合诱导未成熟心肌细胞4，5的成熟。
尽管mpTPP在健康和疾病中的功能意义已经确定，但其确切的分子身份仍然存在争议。mPTP的分子结构和功能研究进展已在别处进行了全面综述12.简言之，目前，mPTP的高导率和低电导状态已被假设为由不同的实体介导12。主要候选者是F1 / F0 ATP合酶（ATP合酶）和腺嘌呤核苷酸转运蛋白（ANT），分别用于高导和低电导模式，分别为12。
尽管对mpTPP的孔隙形成成分的确切身份缺乏共识，但某些关键特征已经详细说明。mPTP的一个公认特征是它受电化学梯度调节，使得IMM的去极化导致孔隙开口13。先前的研究表明，副硫醇基团的氧化还原状态改变了mPTP的电压门控，使得氧化以相对较高的ΔΨs打开孔隙，硫醇基团还原导致闭合mPTP概率为14。然而，蛋白质电压传感器的身份是未知的。
已经确定了调节孔隙开放概率的各种小分子。例如，mPTP可以被钙、无机磷酸盐、脂肪酸和ROS刺激打开，并且可以被腺嘌呤核苷酸（特别是ADP）、镁、质子和CsA5，12抑制。其中一些监管机构的作用机制已经阐明。线粒体钙至少部分通过与ATP合酶15的β亚基结合来触发mPTP开放。ROS可以通过降低其对ADP的亲和力并增强其对亲和力环蛋白D（CypD）的亲和力来激活mpTPP，环蛋白D是研究最好的蛋白质mpTPP激活剂16。无机磷酸盐和脂肪酸活化mpTPP的机制尚不清楚。至于内源性抑制剂，ADP被认为通过在ANT或ATP合酶处结合来抑制mpTPP，而镁通过从其结合位点15，17，18，19中取代钙来发挥其抑制作用。
低 pH 值通过质子化 ATP 合酶 12，20，21 的调节寡霉素敏感性赋予蛋白 （OSCP） 亚基的组氨酸112 来抑制 mPTP 开放。mPTP的典型药物抑制剂CsA通过结合CypD并阻止其与OSCP22，23的关联起作用。先前的工作还表明，各种CoQ类似物与mpTPP相互作用，抑制它或激活它24。在最近的研究中，我们发现由于新生FXS小鼠幼崽的前脑线粒体CoQ缺乏而导致的病理性开放mPTP，过度质子泄漏和低效氧化磷酸化的证据。
用外源性辅酶Q闭合孔阻断了树突状棘的病理性质子泄漏并诱导了形态学成熟25。有趣的是，在相同的动物中，与野生型对照组相比，FXS心肌细胞具有过量的CoQ水平和闭合的mPTP概率26。虽然这些组织特异性CoQ水平差异的原因尚不清楚，但这些发现强调了内源性CoQ可能是mpTPP的关键调节因子的概念。然而，我们的知识存在重大差距，因为辅酶Q介导的mPTP抑制机制仍然未知。
mPTP的调节是细胞信号传导和存活的关键决定因素4。因此，在考虑特定的病理生理机制时，检测线粒体内的mPTP开放是关键。通常，高导电孔隙开口的阈值是使用钙来触发渗透性转变来确定的。这种钙负荷导致膜电位的塌陷，氧化磷酸化的快速解偶联和线粒体肿胀27，28。我们试图开发一种方法来 检测原位 低电导的mPTP开口，而不会诱导它 本身。
该方法利用了mpTPP作为质子泄漏通道的作用。为此，采用Clark型和TPP+ 离子选择性电极分别在泄漏呼吸期间同时测量孤立线粒体中的耗氧量和膜电位29。mPTP打开的阈值由CsA介导的对特定膜电位处质子泄漏的抑制的发作决定。使用这种方法，可以精确地定义在CoQ过量的情况下mPTP的电压门控差异。
Protocol
Representative Results
Discussion
本文介绍了一种评估mpTPP开放概率的方法。具体而言，通过评估CsA抑制对ΔΨs范围内质子泄漏的影响来确定低电导mPTP开路的电压阈值。使用这种技术，我们可以识别FXS小鼠和FVB对照之间mpTPP电压门控的差异，这与它们在组织特异性辅酶Q含量的差异一致。这种方法成功的关键是线粒体在使用前新鲜分离并且质量良好。在分离过程中受损或太老的线粒体将无法产生适当的ΔΨ，并且通常会解耦。请注意?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了以下资助：NIH / NIGMS T32GM008464（K.K.G.），哥伦比亚大学欧文医学中心机会教务长目标麻醉学系（K.K.G.），儿科麻醉学会青年研究奖（K.K.G.）和NIH / NINDS R01NS112706（R.J.L.）。
Materials
| 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | Fisher Scientific | 15630080 | |
| Adapted plunger assembly for pH or ion-selective electrodes for use with OXYT1 | PP systems | 941039 | |
| BD Intramedic PE Tubing, PE 50, 0.023 in. 10 ft. | Fisher Scientific | 14-170-11B | to modify the length of the hamilton synringe as needed |
| Bovine Serum Albumin (BSA). Fatty acid free | Sigma | A7030-10G | |
| Dri-Ref Reference Electrode, 2 mm | World Precision Inst. LLC | DRIREF-2 | |
| Electrode Holder for KWIK-Tips | World Precision Inst. LLC | KWIK-2 | ion selective electrode holder |
| Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) | Sigma | 324626 | |
| FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J | Jackson Laboratory, Bar Harbor, ME | FXS mice, Fmr1 KO | |
| FVB.129P2-Pde6b+ Tyrc-ch/AntJ | Jackson Laboratory, Bar Harbor, ME | FVB mice | |
| Hamilton 80366 Standard Syringes, 10 uL, Cemented-Needle, 6/pk | Cole-Parmer | EW-07938-30 | microsyringe |
| Hamilton 80500 Standard Microliter Syringes, 50 uL, Cemented-Needle | Cole-Parmer | EW-07938-02 | microsyringe |
| Hansatech Instruments Oxytherm+ System (Respiration) Complete | PP systems | OXYTHERM+R | oxygen electrode and software |
| Magnesium Chloride (MgCl2) | Sigma | 1374248 | |
| Mannitol | Sigma | M9546-250G | |
| P1,P5-diadenosine-5′ pentaphosphate pentasodium (AP5A) | Sigma | D4022-10MG | |
| Percoll | Sigma | P1644 | medium for density gradient separation |
| Potassium chloride (KCl) | Sigma | P3911 | |
| Potassium dihydrogen phosphate (KH2PO4) | Sigma | 5.43841 | |
| Sucrose | Sigma | S0389 | |
| TPP+ Electrode Tips (3) | World Precision Inst. LLC | TIPTPP |
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