Lipid modulation of beta-cell calcium channels

描述(由申请人提供)：游离脂肪酸(FFA)的升高导致a细胞内钙离子浓度的增加。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。细胞内钙离子的这种升高可被二氢吡啶抑制，因此依赖于功能上的L型(电压依赖性钙通道)VDCC。我们认为，FFA可引起VDCC的急性反应，增加细胞内钙离子浓度，最终促进胰岛素分泌。我们的荧光共振能量转移分析的初步数据表明，L类型的通道主要在神经内分泌α1D亚基-β细胞中表达，并优先与在其他细胞系统中发现的棕榈酰化的β-2a亚型结合。因此，我们推测FFA介导的反应可能是通过β2a亚基的酰化作用对L类型的α1D-β2a通道产生直接的调制作用。我们建议采用细胞、分子和电生理相结合的方法，研究急性游离脂肪酸诱导的L型通道依赖性细胞内钙升高的分子机制，以及长期暴露于游离脂肪酸后的细胞反应(S)。具体目标是： 1.探讨游离脂肪酸诱导α-细胞内钙升高的细胞机制。我们将通过评估单向钙离子流量和钙含量的变化来量化FFA在β细胞系中诱导的钙处理的变化。在单细胞水平上，我们将评估FFA诱导的钙反应的异质性、链长特异性和浓度依赖性。我们将确定这些作用是由FFA还是其激活形式-长链酰辅酶A发挥的，并区分直接结合和蛋白质酰化。2.确定与FFA诱导效应相关的VDCC分子成分。我们将在表达荧光标记的α1和β亚基不同亚基的COS-7细胞中鉴定L类型的VDCC亚单位(S)，作为FFA作用的靶点，负责DHP抑制的钙升高。我们将产生亚单位(S)的嵌合和定点突变，以定位参与相互作用的残基。我们将研究突变对FFA介导的INS-1细胞胰岛素分泌增加的影响。3.测定游离脂肪酸对VDCC电活动的影响。我们将在β细胞系中进行电生理测试，以测量FFA对通过L类通道的电流的直接影响，并确定FFA增强通过这些VDCC的内向电流的机制。我们将分离FFA对不同L型VDCC的影响，并确定FFA对AIM 2产生的突变通道电流的影响。

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.