Regulation of membrane transport by cardiolipin

心磷脂对膜转运的调节

• 批准号: RGPIN-2019-05368
• 负责人: Hatch, Grant
• 金额: $ 2.33万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=715520
• 关键词: Regulation; membrane; transport; cardiolipin

Cardiolipin (CL) is a key mitochondrial membrane phospholipid required for many cellular functions. Reduction in CL mass by inhibition of its de novo biosynthesis or through loss of the CL remodeling enzyme tafazzin results in reduced mitochondrial function. I hypothesize that CL is required for optimal solute transport and for transcellular transport across biological membranes. Specific Objective 1: Determine the mechanism of how CL regulates solute transport across cell membranes in vitro. We previously demonstrated that reduced CL in blood brain barrier hBMEC/D3 cells increases transport of glucose into and across monolayers of these cells. To further explore how CL regulates solute transport across cell membranes we will reduce CL levels in hCMEC/D3 and examine the transport of solutes and the expression of drug efflux and solute transport proteins. We will culture hCMEC/D3 cells on Transwell® plates and measure apical to basolateral transport of solutes. We will examine how reduced CL modulates transport through protein kinase C epsilon (PKC) in these cells. We anticipate that reduction in CL will result in altered solute transport into and across hBMEC/D3 cells and that PKC activation participates in this process. Preliminary data indicate that creatine transport and mRNA expression of the drug efflux proteins P-glycoprotein and breast cancer resistance protein are reduced when CL is lowered in hCMEC/D3 cells. Specific Objective 2: Determine how CL regulates transport across the blood brain barrier in vivo. We previously demonstrated that knock down of tafazzin in the brain of mice reduces CL. We will temporally examine transport and permeability of radiolabeled mannitol, Evan's blue dye and rhodamine 800 into brain microvessel capillary segments isolated from control and tafazzin knock down (TazKD) mice. We will also use magnetic resonance imaging with Gad-DTPA contrast enhancement to examine transport across the in vivo blood brain barrier. CL levels will then be restored in TazKD mice and the above parameters examined. We anticipate that transport and permeability of radiolabeled mannitol, Evan's blue dye and rhodamine 800 in microvessel capillary segments and Gad-DTPA in vivo permeability will be altered in TazKD mice compared to control animals and that this can be reversed by restoration of normal CL levels. We will confirm the in vivo mechanism by examining how PKC activation is altered in the brain of TazKD mice. The short term research program will identify important membrane transport pathways that are regulated by CL and how changes in CL regulate them. The long-term vision of the research program is to determine how changes in CL can potentially aid in the movement of compounds such as drugs across biological membranes. Given the central role of CL in regulating cellular function these salient biological and biochemical studies are fundamental to our understanding of the basics of membrane transport processes.

心磷脂（CL）是许多细胞功能所需的关键线粒体膜磷脂。通过抑制其从头生物合成或通过CL重塑酶tafazzin的丧失导致CL质量降低，从而导致线粒体功能降低。我假设CL是必需的最佳溶质运输和跨生物膜的跨细胞运输。 具体目标1：确定CL如何在体外调节溶质跨细胞膜转运的机制。我们以前证明，血脑屏障hBMEC/D3细胞中CL减少增加了葡萄糖进入和穿过这些细胞单层的转运。为了进一步探索CL如何调节溶质跨细胞膜转运，我们将降低hCMEC/D3中的CL水平，并检查溶质的转运以及药物外排和溶质转运蛋白的表达。我们将在Transwell®平板上培养hCMEC/D3细胞，并测量溶质的顶侧至基底侧转运。我们将研究如何减少CL调节运输通过蛋白激酶C β（PKC）在这些细胞中。我们预计CL的减少将导致溶质转运进入和穿过hBMEC/D3细胞的改变，并且PKC活化参与了这一过程。初步数据表明，当CL在hCMEC/D3细胞中降低时，肌酸转运和药物外排蛋白P-糖蛋白和乳腺癌耐药蛋白的mRNA表达降低。 具体目标2：确定CL如何调节体内血脑屏障转运。我们先前证明，敲低小鼠脑中的tafazzin可降低CL。我们将暂时检查运输和渗透性的放射性标记的甘露醇，伊文思蓝染料和罗丹明800到脑微血管毛细血管段分离对照和tafazzin敲低（TazKD）小鼠。我们还将使用Gad-DTPA对比增强的磁共振成像来检查穿过体内血脑屏障的运输。然后在TazKD小鼠中恢复CL水平，并检查上述参数。我们预计，与对照动物相比，TazKD小鼠中放射性标记的甘露醇、伊文思蓝染料和罗丹明800在微血管毛细血管段中的转运和渗透性以及Gad-DTPA在体内的渗透性将发生改变，并且这可以通过恢复正常的CL水平来逆转。我们将通过研究TazKD小鼠大脑中PKC激活如何改变来证实体内机制。 短期研究计划将确定重要的膜转运途径，由CL和CL的变化如何调节它们。该研究计划的长期愿景是确定CL的变化如何可能有助于化合物（如药物）穿过生物膜的运动。鉴于CL在调节细胞功能中的核心作用，这些突出的生物学和生物化学研究对于我们理解膜转运过程的基础是至关重要的。