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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738227
• 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 Transwellr plates and measure apical to basolateral transport of solutes. We will examine how reduced CL modulates transport through protein kinase C epsilon (PKCe) in these cells. We anticipate that reduction in CL will result in altered solute transport into and across hBMEC/D3 cells and that PKCe 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 PKCe 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)是许多细胞功能所必需的关键线粒体膜磷脂。通过抑制CL的从头生物合成或失去CL重塑酶Tafazzin，CL质量减少会导致线粒体功能降低。我假设CL是最佳溶质运输和跨细胞生物膜运输所必需的。具体目标1：确定CL在体外调节溶质跨细胞膜转运的机制。我们先前证明，血脑屏障hBMEC/D3细胞中CL的减少增加了葡萄糖在这些细胞单层内和跨单层的运输。为了进一步探讨CL如何调节溶质跨细胞膜的转运，我们将降低hCMEC/D3中CL的水平，并检测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Trswellr平板上培养hCMEC/D3细胞，并测量溶质从顶端到底侧的转运。我们将研究减少的CL如何通过蛋白激酶C epsilon(PKCE)在这些细胞中调节运输。我们预计CL的减少将导致溶质进入和穿过hBMEC/D3细胞的运输发生改变，PKCE激活参与了这一过程。初步数据表明，当CL降低时，hCMEC/D3细胞的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表达减少。具体目标2：确定CL-1在体内如何调节血脑屏障的转运。我们先前已经证明，在小鼠的大脑中击倒他法津可以降低CL。我们将在时间上检测放射性标记甘露醇、伊文氏蓝染料和罗丹明800对对照组和他法津击倒(Tazazzin)小鼠脑微血管毛细血管的转运和渗透性。我们还将使用磁共振成像和Gad-DTPA对比度增强来检查体内血脑屏障的运输。然后恢复TazKD小鼠的CL水平，并检测上述参数。我们预计，与对照动物相比，TazKD小鼠体内放射性标记甘露醇、伊文氏蓝染料和罗丹明800在微血管毛细血管段的转运和通透性以及Gad-DTPA的通透性将发生改变，这种变化可以通过恢复正常的CL水平来逆转。我们将通过检测TazKD小鼠大脑中PKCE的激活是如何改变的来确认体内的机制。短期的研究计划将确定CL调控的重要膜转运途径，以及CL的变化是如何调控它们的。这项研究计划的长期愿景是确定CL的变化如何潜在地帮助药物等化合物在生物膜上的移动。鉴于CL在调节细胞膜功能中的核心作用，这些重要的生物学和生化研究对于我们理解膜转运过程的基础是基本的。