STRUCTURE OF MITOCHONDRIAL K+ CHANNELS

线粒体ATP依赖性钾通道(mitoK[ATP])和线粒体钙依赖性钾通道(MitoKca)在缺血预适应过程中起重要作用。在这里，我们试图确定mitoKATp和mitoKca的分子同一性。我们的方法使用了由来已久的、以分析为导向的蛋白质纯化原则，并结合了新的敏感蛋白质组学工具，包括二维凝胶电泳(2DGE)和质谱仪(MS)。为了克服细胞内这些通道的稀缺，我们将从猪的肝脏中分离出多达10克的线粒体，比以前在这类研究中使用的线粒体多几个数量级。 纯化将涉及多轮层析，包括利用这些K+通道的生物特异性的亲和层析。在mitoK[ATP]的情况下，我们将利用其与ATP的相互作用，而对于mitoKca，我们将利用其与蝎子毒素毒素的高亲和力相互作用。合并策略的稳健性将源于冗余分析的使用。首先，巨型蛋白脂质体中钾通道电流的功能重构将为钾通道活性提供最终的评估。其次，我们还将采用针对mitoK[ATP]和mitoKca的检测方法。我们识别K+通道富集组分中蛋白质的能力将由灵敏的蛋白质组学工具--多肽-质量指纹图谱(PMF)和串联质谱学(MS/MS)确定。或者，如果产量足够，将通过Edman降解获得蛋白质序列。结构性 初级氨基酸序列或PMF-MS/MS数据形式的信息随后将指导这些通道的克隆。新鉴定的通道的基本性质，包括药理学特异性，将使用膜片钳分析来测量巨型蛋白脂质体中的K+选择电流。通过蛋白质交联实验，研究分子结构与天然相对分子质量、亚基化学计量比和亚基间接触的关系。最后，我们将确定任何翻译后修饰或K+通道相互作用的蛋白质。

Two intracellular channels, the mitochondrial ATP-dependent potassium channel (mitoK[ATP]) and the mitochondrial Ca2+-dependent potassium channel (mitoKca), figure prominently in the process of ischemic preconditioning. Here we seek to determine the molecular identities of mitoKATp and mitoKca. Our approach employs time-honored, assay-driven, protein purification principles, in conjunction with new sensitive proteomic tools that include 2-dimensional gel electrophoresis (2DGE) and mass spectrometry (MS). To overcome the scarcity of these channels within the cell, we will isolate up to 10 g of mitochondria from pig liver, several orders of magnitude more mitochondria than have been used in previous studies of this type. Purification will involve multiple rounds of chromatography, including affinity chromatography to harness the biospecificity of these K+-channels. In the case of mitoK[ATP], we will exploit its interaction with ATP, whereas for mitoKca, we will exploit its high-affinity interaction with the scorpion venom protein, iberiotoxin. The robustness of the pooling strategy will stem from the use of redundant assays. Firstly, functional reconstitution of K+-channel current in giant proteoliposomes will provide the ultimate assessment of potassium channel activity. Secondly, we will also employ assays that are specific to mitoK[ATP] and mitoKca. Our ability to identify proteins in K+-channel-enriched fractions will be determined by the sensitive proteomic tools of peptide-mass fingerprinting (PMF) and tandem mass-spectrometry (MS/MS). Alternatively, if yields are sufficient, protein sequence will be obtained by Edman degradation. Structural information, in the form of primary amino acid sequence or PMF-MS/MS data, will subsequently guide the cloning of these channels. The fundamental properties of the newly-identified channels, including pharmacological specificity, will be assessed using patch-clamp analysis to measure K+-selective current in giant proteoliposomes. Molecular architecture will be studied with respect to native molecular weight, subunit stoichiometry and intersubunit contacts, through protein crosslinking experiments. Finally, we will identify any post-translational modifications or K+-channel-interacting proteins.