Lipid modulation of beta-cell calcium channels

β 细胞钙通道的脂质调节

• 批准号: 6768542
• 负责人: LINA M MOITOSO DE VARGAS
• 金额: $ 23.3万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6768542
• 关键词: acyl coA; acylation; calcium channel; calcium flux; cell line; fluorescence microscopy; fluorescence resonance energy transfer; free fatty acids; immunoprecipitation; insulin; lipids; pancreatic islet function; pancreatic islets; site directed mutagenesis; voltage /patch clamp

DESCRIPTION (provided by applicant): Elevation of free fatty acids (FFA) results in an increase in the intracellular Ca2+ concentration of the a-cell. This effect does not appear to involve alterations in membrane potential, but is consistent with the augmented insulin release observed with acute exposure to FFA. This rise in intracellular Ca2+ was inhibited by dihydropyridines and thus depended on functional L-type (voltage-dependent calcium channels) VDCC. We propose that FFA elicit an acute response of the VDCC, increasing the intracellular Ca2+ concentration and ultimately insulin secretion. Our preliminary data from the fluorescence resonance energy transfer analyses indicate that the L-type channel predominantly expressed in beta-cells, the neuroendocrine alpha1D subunit, preferentially associates with a beta2a isoform found to be palmitoylated in other cell systems. Thus, we hypothesize that the FFA-mediated response may be exerted by a direct modulatory effect on the L-type alpha1D-beta2a channel through acylation of the beta2a subunit. We propose to discern the molecular mechanisms underlying the acute FFA-induced, L-type channel-dependent increase in intracellular Ca2+, as well as the cellular response(s) following long term exposure to FFA, using a combination of cellular, molecular and electrophysiological approaches. The specific aims are: 1. To determine the cellular mechanisms underlying the FFA-induced Ca2+ rise in a-cells. We will quantitate the FFA-induced changes in Ca2+ handling in beta-cell lines by assessing changes in unidirectional Ca2+ fluxes and calcium content. At the single cell level we will evaluate the heterogeneity, chain length specificity and concentration dependence of the Ca2+ response induced by FFA. We will establish whether these effects are exerted by FFA or its activated form, long chain acyl-CoA and distinguish between direct binding and protein acylation. 2. To determine the VDCC molecular components responsible for the FFA-induced effects. We will identify in COS-7 cells expressing various combinations of fluorescent-labeled alpha1 and beta subunit isoforms the L-type VDCC subunit(s) that, as the target of FFA action, is responsible for the DHP-inhibitable Ca2+ rise. We will generate chimeric and site-directed mutants of the subunit(s) to pinpoint the residues involved in the interaction. We will examine the effects of the mutations on FFA-mediated augmentation of insulin secretion in INS-1 cells. 3. To determine the effects of FFA on the electrical activity of VDCC. We will perform electrophysiological assays in beta-cell lines to measure the direct effects of FFA on currents through L-type channels and to determine the mechanism by which FFA enhance inward currents through those VDCC. We will separate the effects of FFA on different L-type VDCC and determine the effects of FFA on currents through mutant channels generated in Aim 2.

描述（由申请人提供）：游离脂肪酸（FFA）的升高导致α-细胞的细胞内Ca 2+浓度增加。这种效应似乎不涉及膜电位的改变，但与急性暴露于FFA时观察到的胰岛素释放增加一致。细胞内Ca 2+的这种升高被二氢吡啶类抑制，因此依赖于功能性L型（电压依赖性钙通道）VDCC。我们建议FFA引起VDCC的急性反应，增加细胞内Ca 2+浓度并最终增加胰岛素分泌。我们从荧光共振能量转移分析的初步数据表明，主要在β细胞中表达的L型通道，神经内分泌α 1D亚基，优先与β 2a亚型在其他细胞系统中被棕榈酰化。因此，我们假设FFA介导的反应可能是通过β 2a亚基的酰化对L型α 1D-β 2a通道的直接调节作用而产生的。我们建议使用细胞、分子和电生理学方法的组合来辨别急性FFA诱导的L型通道依赖性细胞内Ca 2+增加的分子机制，以及长期暴露于FFA后的细胞反应。具体目标是： 1.确定FFA诱导的α-细胞内Ca 2+升高的细胞机制。我们将通过评估单向Ca 2+通量和钙含量的变化来定量FFA诱导的β细胞系中Ca 2+处理的变化。在单细胞水平，我们将评估的异质性，链长特异性和浓度依赖性的钙离子反应诱导FFA。我们将确定这些作用是否由FFA或其活化形式长链酰基辅酶A产生，并区分直接结合和蛋白酰化。2.确定导致FFA诱导效应的VDCC分子组分。我们将在表达荧光标记的α 1和β亚基同种型的各种组合的COS-7细胞中鉴定L型VDCC亚基，作为FFA作用的靶点，其负责DHP可降解的Ca 2+升高。我们将产生亚基的嵌合和定点突变体，以确定参与相互作用的残基。我们将研究这些突变对FFA介导的INS-1细胞胰岛素分泌增强的影响。3.探讨游离脂肪酸（FFA）对VDCC电活动的影响。我们将在β细胞系中进行电生理学测定，以测量FFA对通过L型通道的电流的直接影响，并确定FFA通过这些VDCC增强内向电流的机制。我们将分离FFA对不同L型VDCC的影响，并确定FFA对通过Aim 2中产生的突变通道的电流的影响。