Name: f1fo atpase vesicle preparation and technique for performing patch clamp recordings of submitochondrial vesicle membranes
Uploaded: 2013-05-04T14:00:00
Description: Watch this Scientific Journal Video about F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes at JoVE.com

1。脑线粒体隔离（ 改编自布朗MR等。9） <ol><li>牺牲大鼠批准的机构动物护理和使用委员会（IACUC）的使用方法。 </li><li>切头斩首的动物，切开皮肤，暴露颅骨。 </li><li>轻轻地打开颅骨切割用剪刀或咬骨钳。取出大脑。 </li><li>百果精细小脑脑无隔离缓冲（ 见表1），并将其转移到5毫升的玻璃/聚四氟乙烯匀浆器（设备列表）。 </li><li>轻轻均质组织10次（无气泡），约5...

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	<li>Cheng, W. C., Berman, S. B., Ivanovska, I., Jonas, E. A., Lee, S. J., Chen, Y., Kaczmarek, L. K., Pineda, F., Hardwick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+factors+with+dual+roles+in+death+and+survival.">Mitochondrial factors with dual roles in death and survival.</a> Oncogene. 25, 4697-4705 (2006).</li><li>Duchen, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+calcium+in+health+and+disease.">Mitochondria and calcium in health and disease.</a> Cell Calcium. 44, 1-5 (2008).</li><li>Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+mitochondrial+membrane+permeability+in+pathogenesis,+autophagy+and+control+of+metabolism.">Modulation of mitochondrial membrane permeability in pathogenesis, autophagy and control of metabolism.</a> J. Gastroenterol. Hepatol. 22, S31-S37 (2007).</li><li>Cox, G. B., Jans, D. A., Fimmel, A. L., Gibson, F., Hatch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothesis.+The+mechanism+of+ATP+synthase.+Conformational+change+by+rotation+of+the+beta-subunit.">Hypothesis. The mechanism of ATP synthase. Conformational change by rotation of the beta-subunit.</a> Biochim. Biophys. Acta. 768, 201-208 (1984).</li><li>Cox, G. B., Fimmel, A. L., Gibson, F., Hatch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+ATP+synthase:+a+reassessment+of+the+functions+of+the+b+and+a+subunits.">The mechanism of ATP synthase: a reassessment of the functions of the b and a subunits.</a> Biochim. Biophys. Acta. 849, 62-69 (1986).</li><li>Cory, S., Huang, D. C., Adams, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bcl-2+family:+roles+in+cell+survival+and+oncogenesis.">The Bcl-2 family: roles in cell survival and oncogenesis.</a> Oncogene. 22, 8590-8607 (2003).</li><li>Vander Heiden, M. G., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+proteins:+regulators+of+apoptosis+or+of+mitochondrial+homeostasis.">Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis.</a> Nat. Cell Biol. 1, 209-216 (1999).</li><li>Alavian, K. N., Li, H., Collis, L., Bonanni, L., Zeng, L., Sacchetti, S., Lazrove, E., Nabili, P., Flaherty, B., Graham, M., Chen, Y., Messerli, S. M., Mariggio, M. A., Rahner, C., McNay, E., Shore, G. C., Smith, P. J. S., Hardwick, J. M., Jonas, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-xL+regulates+metabolic+efficiency+of+neurons+through+interaction+with+the+mitochondrial+F1FO+ATP+synthase.">Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase.</a> Nat. Cell Biol. 13 (10), 1224-1233 (2011).</li><li>Brown, M. R., Sullivan, P. G., Dorenbos, K. A., Modafferi, E. A., Geddes, J. W., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+disruption+of+synaptoneurosomes:+an+alternative+method+to+isolate+brain+mitochondria.">Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria.</a> Journal of Neuroscience Methods. 137, 299-303 (2004).</li><li>Chan, T. L., Greenawalt, J. W., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+ultrastructural+properties+of+a+mitochondrial+inner+membrane+fraction+deficient+in+outer+membrane+and+matrix+activities.">Biochemical and ultrastructural properties of a mitochondrial inner membrane fraction deficient in outer membrane and matrix activities.</a> J. Cell Biol. 45 (2), 291-305 (1970).</li><li>Young, H. K. o., Delannoy, M., Hullihen, J., Chiu, W., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ATP+Synthasomes.">Mitochondrial ATP Synthasomes.</a> J. Biol. Chem. 278 (14), 12305-12309 (2003).</li></ol>

描述的方法，本文纯线粒体使隔离结束时，步骤1和步骤的submitochondrial囊泡（SMVS）在步骤2之后从全脑的细胞此方法phenotypes.SMVspurified的没有区别，基本上不含污染的其它亚细胞器所示图1和我们以前的工作（Alavian KN 等 。8），并保留其结构和功能的完整性，在冷冻之前。冻融后，分离线粒体或SMVS用于录音和生化检测，但没有呼吸的研究。 此外，此?...

<table border="1">
 <tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Potter-Elvehjem Tissue Grinder withPTFEPestle</td>
 <td>Krackeler Scientific, Inc.</td>
 <td>1-7725T-5 </td>
 </tr>
 <tr>
 <td>Eppendorf Centrifuge 5424 </td>
 <td>Eppendorf </td>
 <td>5424 000.410</td>
 </tr>
 <tr>
 <td>4639 Cell Disruption Vessel</td>
 <td>Parr Instrument Company</td>
 <td>4639</td>
 </tr>
 <tr>
 <td>Ficoll</td>
 <td>Sigma-Aldrich</td>
 <td>F5415</td>
 </tr>
 <tr>
 <td>Polycarbonate centrifuge tubes</td>
 <td>Beckman Coulter</td>
 <td>P20314</td>
 </tr>
 <tr>
 <td>SW-50.1 rotor </td>
 <td>Beckman Coulter</td>
 <td></td>
 </tr>
 <tr>
 <td>L8-70M Ultracentrifuge</td>
 <td>Beckman Coulter</td>
 <td></td>
 </tr>
 <tr>
 <td>Digitonin</td>
 <td>Sigma-Aldrich</td>
 <td>D5628 </td>
 </tr>
 <tr>
 <td>Lubrol PX (C12E9) </td>
 <td>Calbiochem</td>
 <td>205534</td>
 </tr>
 <tr>
 <td>Axopatch 200B </td>
 <td>Axon Instruments </td>
 <td></td>
 </tr>
 <tr>
 <td>Digidata 1440A </td>
 <td>Molecular Device</td>
 <td></td>
 </tr>
 <tr>
 <td>pClamp10.0</td>
 <td>Molecular Device</td>
 <td></td>
 </tr>
 <tr>
 <td>Manipulator</td>
 <td>Sutter Instrument</td>
 <td></td>
 </tr>
 <tr>
 <td>Borosilicate glass capillary</td>
 <td>World Precision Instruments</td>
 <td>1308325</td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller Model P-87</td>
 <td>Sutter Instrument</td>
 <td></td>
 </tr>
 </tbody>
</table>

f1fo atpase vesicle preparation and technique for performing patch clamp recordings of submitochondrial vesicle membranes

线粒体都参与许多重要的细胞功能，包括新陈代谢，生存，发展和钙信号2。的两个最重要的线粒体功能相关的高效生产的ATP，通过氧化磷酸化作用，细胞的能量货币，以及调解的程序性细胞死亡的信号3。 主要负责产生ATP的酶F1FO ATP合成酶，也称为ATP合酶4-5。近年来，线粒体的凋亡细胞和坏死细胞死亡的作用已经受到人们的关注。细胞凋亡，如Bax的BCL-2家族蛋白进入线粒体外膜，低聚和通透的外膜，促凋亡因子释放到细胞质6。在经典的坏死性细胞死亡中，例如，所产生的局部缺血或神经元的兴奋性毒性，LARGE，监管不力，在基体中的钙有助于增加内膜毛孔，线粒体通透性转换孔或mPTP的开放。此去极化内膜，促进外膜破裂，释放促凋亡因子和代谢功能紊乱，导致渗透压的变化。许多蛋白质，包括了Bcl-xL 7与F1FO ATP合成酶，调节其功能。 Bcl-xL的直接交互的F1FO ATP合酶β亚基，这种相互作用降低泄漏电导在F1FOATPasecomplex内的，增加的H +净运输期间F1FO F1FO ATPase活性8，从而增加线粒体效率。要研究ATP合成酶的活性和调制，我们孤立从啮齿动物脑submitochondrial囊泡（SMVS）含有F1FO ATP酶的。 SMVS保留F1FO ATP酶的结构和功能完整性所示在Alavian 等 。在这里，我们描述了一种方法，我们已经成功地用于隔离SMVS大鼠脑和我们划定膜片钳技术分析通道活性（离子泄漏电导）SMVS。

我们的协议的第一步骤中可以分离纯化的线粒体，如在图1中所示的Western印迹。在图2中示出一个脑源性submitochondrial的囊泡补丁记录的一个例子。使用内而外的补丁配置，我们展示了调制由ATP通道的活性。控制（CTL）录音（左）显示多导通道活动的平均电导为600 PS的峰值。立刻被减少后1毫米ATP洗澡。三磷酸腺苷的整体效果是SMVS的补丁以浓度依赖的方式减少的电导。要测量...

Watch this Scientific Journal Video about F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes at JoVE.com

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

一种方法隔离submitochondrial的富集大鼠脑F1FO ATP合酶复合囊泡。这些囊泡允许F1FO ATP酶的活性配合物及其调制使用膜片钳记录技术的研究。

<sub&gt; 1</sub&gt; F<sub&gt; O</subATP酶微囊的制备和表演膜片钳记录的Submitochondrial泡膜技术

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Research

JoVE Journal

Neuroscience

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

NRCC的膜片钳芯片上细胞的培养和电

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...