Regulation of membrane transport by cardiolipin

心磷脂（Cardiolipin， CL）是ATP生成所需的关键线粒体膜磷脂。合成后，它与亚油酸在他法津酶的作用下被改造成四烷油酰心磷脂（L4-CL）。CL的减少和/或其脂肪酸组成的改变导致生成ATP的能力降低。我们假设CL的适当含量和脂肪酸组成是最佳溶质运输和跨生物膜的细胞运输所必需的。特定目的1：确定心磷脂是否调节溶质和药物在体外跨细胞膜的转运。通过siRNA敲低CL合酶（hCLS1），可以降低hCMEC/D3细胞中的CL水平。测定对照组和低CL细胞的线粒体能量代谢，检测溶质、药物外排转运体、脂肪酸转运体以及紧密连接蛋白和炎症标志物的水平和转运能力。膜通透性和吸收放射性标记脱氧葡萄糖，放射性标记脂肪酸和罗丹明800将被检查。我们预计，随着CL水平的降低，hCMEC/D3细胞的线粒体功能（包括基础耗氧量、糖酵解率、呼吸能力和ATP周转率）将降低，hCMEC/D3细胞中CL水平的降低将导致脂肪酸、葡萄糖、肌酸和罗丹明800转运到hCMEC/D3细胞中的减少。我们将在Transwell®板上培养hCMEC/D3细胞，并测量放射标记的葡萄糖、肌酸、油酸和罗丹明800的根尖向基底侧转运。预计在Transwell®板上培养的hCMEC/D3细胞中CL的减少将导致线粒体功能降低，脂肪酸、葡萄糖、肌酸和罗丹明800转运到细胞中减少。通过表达hCLS1来恢复细胞中的CL水平，这些细胞中的CL水平因hCLS1敲除而降低，并检查了所有上述参数。此外，作为推论实验，在hCMEC/D3细胞中表达hCLS1会升高CL水平，并检测了上述参数。我们预计，适当的CL含量是溶质和药物运输过程的适当功能所必需的。特定目的2：确定心磷脂是否在体内调节溶质和药物通过血脑屏障（BBB）的运输。小鼠体内的L4-CL水平将通过降低他法嗪和放射性标记甘露醇的转运和通透性而降低，Evan的蓝色染料和罗丹明800的通透性将暂时从这些动物和对照动物中测定。将从大脑中分离微血管毛细血管段并确定线粒体功能。此外，磁共振成像与Gad-DTPA对比增强将用于检查血脑屏障的运输在体内。他法嗪被敲除后，小鼠的L4-CL水平将恢复，并检查上述参数。我们预计，微血管毛细血管段的线粒体功能、放射性标记甘露醇和罗丹明800的运输和通透性以及Gad-DTPA的通透性将因他法嗪敲低而降低，而这可以通过恢复正常的L4-CL水平来逆转。拟议的研究将确定重要的运输/调节途径，可以通过线粒体功能的改变来调节CL。这些研究将创建一个新的研究项目，具有表征溶质和药物膜运输的长期愿景，并确定通过CL介导的线粒体功能如何影响这一过程，并可能有助于调节膜功能的药物开发。这些生物学研究在药物开发中具有重要的基础意义。

Cardiolipin (CL) is a key mitochondrial membrane phospholipid required for the generation of ATP. Upon its synthesis CL is remodeled with linoleic acid to form tetralinoleoyl-cardiolipin (L4-CL) by the enzyme tafazzin. Reduction in CL and/or alteration in its fatty acid composition results in reduced ability to generate ATP. We hypothesize that the appropriate content and fatty acid composition of CL is required for optimal solute transport and transcellular transport across biological membranes. Specific Objective 1: Determine if cardiolipin regulates solute and drug transport across cell membranes in vitro. CL levels in hCMEC/D3 cells will be lowered using siRNA knock down of CL synthase (hCLS1). Mitochondrial energy metabolism in control and cells with reduced CL will be determined and the level and transport ability of solute, drug efflux transporters, fatty acid transporters as well as tight junction proteins and inflammatory markers will be examined. The membrane permeability and uptake of radiolabeled deoxyglucose, radiolabeled fatty acid and rhodamine 800 will be examined. We anticipate that mitochondrial function including basal oxygen consumption, glycolysis rate, respiratory capacity and ATP turnover will be lower in hCMEC/D3 cells with reduced CL levels and that reduction in CL in hCMEC/D3 cells will result in reduced fatty acid, glucose, creatine and rhodamine 800 transport into hCMEC/D3 cells. We will culture hCMEC/D3 cells on Transwell® plates and measure apical to basolateral transport of radiolabeled glucose, creatine, oleate, and transport of rhodamine 800. It is anticipated that reduction in CL in hCMEC/D3 cells cultured on Transwell® plates will result in reduced mitochondrial function, reduced fatty acid, glucose, creatine and rhodamine 800 transport into cells. CL levels will be restored by expressing hCLS1 in cells in which CL levels were reduced by hCLS1 knock down and all of the above parameters examined. In addition, as corollary experiments, CL levels will be elevated by expressing hCLS1 in hCMEC/D3 cells and the above parameters examined. We anticipate that the appropriate content of CL is required for proper function of solute and drug transport processes. Specific Objective 2: Determine if cardiolipin regulates solute and drug transport across the blood brain barrier (BBB) in vivo. Whole body L4-CL levels will be reduced by knock down of tafazzin in mice and the transport and permeability of radiolabeled mannitol, and Evan’s blue dye and rhodamine 800 permeability will be temporally determined from these and control animals. Microvessel capillary segments will be isolated from brains and mitochondrial function determined. In addition, magnetic resonance imaging with Gad-DTPA contrast enhancement will be used to examine transport across the BBB in vivo. L4-CL levels will be restored in mice in which tafazzin was knocked down and the above parameters examined. We anticipate that mitochondrial function in microvessel capillary segments, transport and permeability of radiolabeled mannitol and rhodamine 800 and Gad-DTPA permeability will be reduced by tafazzin knock down and that this can be reversed by restoration of normal L4-CL levels.The proposed studies will identify important transport/modulation pathways that can be regulated by mitochondrial function through changes in CL. These studies will create a new research program with the long-term vision of characterizing solute and drug membrane transport and determining how mitochondrial function mediated through CL can influence this and potentially aid in drug development for regulation of membrane function. These biological studies are of fundamental importance in drug development.