A Collaborative Project: Molecular Studies of A Mitochondrial Ion Channel

;​R o o t E n t y F p +- C o m p o b j b W o r d d o c u m e n t ;0 0 0 0 0 0 0 0 *- *- 4 @ 5 6 7 8 9；= ?F Microsoft Word 6.0文档MSWordDoc。6;​9513439氧化磷酸化与离子和代谢物穿过包围线粒体的两层膜的通量密切相关；离子流动的一种途径是通过离子通道。外膜和内外膜之间的接触部位的调节作用才刚刚开始被认识和理解。这一建议的重点是能量耦合内膜及其主要通道活性，一个多电导通道，MCC。门控通道是通过膜的可调节的分子孔，其开放受生理刺激（如电压和胞内钙）的调节。本提案的主要目的是确定MCC的生理和分子基础。这些知识对于在分子水平上理解控制内膜通透性的因素是必要的。此外，这一信息可能为在变化的条件下（例如在运动或缺血期间），所有五个内膜通道与线粒体能量产生调节相互作用的机制提供见解。该提案有几个与其总体目标相关的目标，这些目标将通过电生理和生物物理技术的结合来实现。其他人已经提出通道参与细胞质中合成的线粒体蛋白质的易位，我们有证据表明MCC可能在蛋白质输入中发挥作用。我们将主要使用膜片钳技术检查针对异寡聚进口装置中假定的内膜通道部分的抗体和几种突变体对MCC活性的影响。同时，MCC活性将通过其他研究，如电压依赖性阴离子通道和腺嘌呤核苷酸转位器，在涉及MCC活性的蛋白质的缺失突变体中进行检测。这两方面的研究将继续进行，直到确定MCC的分子基础和生理作用。纯化的MCC成分的生物物理特性将包括与有丝体细胞中的MCC的电生理比较以及(-螺旋和（-片）含量的圆二色性测定。最后，将研究MCC的结构/功能及其与内外膜接触部位的关系。*** ;Oh +' 0 $ H l D H R:\WWUSER\TEMPLATE\NORMAL。DOT (S)：我是说，我是说，我是说，我是说，我是说，我是说，我是说，我是说，我是说9513439 Thomasina M Osborne @ *- @ @ *- @ f# Microsoft Word 6.0 2;e = e A；一个 j j j j j 你1 # 7 W T u j u j j j氧化磷酸化与离子和代谢物在线粒体外膜上的流动密切相关；离子流动的一种途径是通过离子通道。外膜和内外膜之间的接触部位的调节作用才刚刚开始被认识和理解。这一建议的重点是能量耦合内膜及其主要通道活性，一个多电导通道，MCC。门控通道是通过膜的可调节的分子孔径

; R o o t E n t r y F p+- C o m p O b j b W o r d D o c u m e n t ; O b j e c t P o o l *- *- 4 @ 5 6 7 8 9 : ; = ? F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; 9513439 Kinnally Oxidative phosphorylation is intimately connected to the flux of ions and metabolites across the two membranes enclosing mitochondria; one pathway for ion flux is through ion channels. The regulatory role of the outer membrane and of the contact sites between the inner an outer membranes is just beginning to be recognized and understood. This proposal focusses on the energy-coupling inner membrane and its predominant channel activity, a multiple conductance channel, MCC. Gated channels are adjustable molecular apertures through membranes whose opening is regulated by physiological stimuli such as voltage and the intracellular calcium in the case of MCC. The main goal of this proposal is to determine the physiological and molecular basis of MCC. This knowledge is necessary for understanding factors controlling the inner membrane permeability at the molecular level. Furthermore, this information may provide insight into the mechanisms by which all five inner membrane channels interact with the regulation of energy production in mitochondria under changing conditions, e.g. durin g exercise or ischemia. This proposal has several objectives related to its overall goal, which are to be achieved through a combination of electrophysiological and biophysical techniques. It has been proposed by others that channels are involved in the translocation of mitochondrial proteins synthesized in the cytoplasm and we have evidence MCC may play a role in protein import. Antibodies to and several mutants of the putative inner membrane channel portion of the hetero-oligomeric import apparatus will be examined for effects on MCC activity primarily using patch-clamp techniques. At the same time, MCC activity will be examined in deletion mutants of proteins implicated in MCC activity by other studies, e.g. voltage dependent anion channel and adenine nucleotide translocator. These two lines of investigation will continue until MCC s molecular basis and physiological role are determined. A biophysical characterization of the purified MCC constituent(s) will include electrophysiological comparisons to MCC in mitoplasts and circular dichroism determination of (-helix and (-sheet content. Finally, structure/function studies will be made of MCC and its relationship with contact sites between the inner and outer membranes. *** ; Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT S u m m a r y I n f o r m a t i o n ( 4 9513439 Thomasina M Osborne Thomasina M Osborne @ *- @ @ *- @ F # Microsoft Word 6.0 2 ; e = e A ; A j j j j j j j u 1 # 7 W T A u j u j j j j ~ j j j j j 9513439 Kinnally Oxidative phosphorylation is intimately connected to the flux of ions and metabolites across the two membranes enclosing mitochondria; one pathway for ion flux is through ion channels. The regulatory role of the outer membrane and of the contact sites between the inner an outer membranes is just beginning to be recognized and understood. This proposal focusses on the energy-coupling inner membrane and its predominant channel activity, a multiple conductance channel, MCC. Gated channels are adjustable molecular apertures through membranes whose op