CALMODULIN FUNCTION IN THE CELL

细胞中的钙调蛋白功能

• 批准号: 6177604
• 负责人: ANTHONY J PERSECHINI
• 金额: $ 19.14万
• 依托单位: UNIVERSITY OF MISSOURI KANSAS CITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177604
• 关键词: adenylate cyclase; calcium binding protein; calcium flux; calcium ion; calmodulin; cell line; enzyme activity; enzyme induction /repression; fluorescent dye /probe; membrane proteins; protein kinase C; transfection

When it binds Ca2+, calmodulin (CaM) activates an enormous array of different enzymes with critical functions in the cell. Our preliminary results indicate the maximum global free concentration of Ca2+-CaM in cells is approximately 30 nM, and the total concentration of CaM is well below the concentration of CaM-binding proteins. While many CaM-dependent enzymes bind Ca2+-CaM with dissociation constants of approximately 1 nM, some have affinities hundreds of times lower. Among this group are the CaM-dependent adenylate cyclases, which are robustly activated by Ca2+ in cells. This is not consistent with the low global free concentrations of Ca2+-CaM that we have measured. However, conditions that produce local increases in the free Ca2+ concentration at the plasma membrane appear to be required to significantly activate the cyclases in several cell types. And they co-localize in the brain with neuromodulin or neurogranin, membrane-associated proteins thought to function as CaM "sinks". We have therefore hypothesized the existence of two mechanisms that enhance the free Ca2+-CaM concentrations at the plasma membrane to produce the observed activation of the cyclases: (1) Diffusional recruitment of CaM due to local elevations in Ca2+, and (2) Concentration of CaM at the plasma membrane by CaM sink proteins. We propose to test this hypothesis by determining how the free concentrations of Ca2+-CaM produced at the plasma membrane are affected by local elevations in the free Ca2+ concentration and expression of neuromodulin, and how this relates to changes in the activity an expressed CaM-dependent cyclase activity (AC1). We also,,will investigate whether PKC- catalyzed phosphorylation of neuromodulin, which abolishes its CaM sink function, can dynamically control free Ca2+-CaM concentrations and cyclase activity. Given the limiting amount of CaM in the cell, it is clearly of general importance to understand how it is distributed among CaM-dependent enzymes, and how this impacts cellular function. This proposal emphasizes factors that enhance the free Ca2+-CaM concentrations at the plasma membrane and control the activities of CaM-dependent cyclases, as well as other low affinity CaM-dependent enzymes. The cyclases are a particularly important class of low affinity CaM targets, having been specifically been implicated in secretion and steroidogenesis in parotid acinar and adrenal glomerulosa cells, and in some forms of learning.

当它结合Ca2+时，钙调蛋白（CAM）激活具有细胞中关键功能的不同酶的巨大阵列。我们的初步结果表明，细胞中Ca2+-CAM的最大全局游离浓度约为30 nm，CAM的总浓度远低于CAM结合蛋白的浓度。虽然许多CAM依赖性酶将CA2+-CAM与约1 nm的解离常数结合，但有些较低的亲和力降低了数百倍。该组中有CAM依赖性的腺苷酸环化酶，这些腺苷酸环化酶在细胞中被Ca2+强化。这与我们测量的CA2+-CAM的全球游离浓度低不一致。但是，在质膜的游离Ca2+浓度中产生局部增加的条件似乎是需要显着激活几种细胞类型中的循环酶的。它们与神经统治或神经素蛋白共定位在大脑中，膜相关的蛋白被认为是CAM“下沉”。因此，我们假设存在两种机制，这些机制可以增强质膜处的游离Ca2+-CAM浓度，从而产生观察到的循环酶的激活：（1）由于Ca2+中局部升高引起的CAM的扩散，并且（（2）CAM浓度的CAM浓度在凸轮膜上通过CAM膜通过CAMMMBRANE通过凸轮膜下的蛋白。我们建议通过确定在质膜上产生的Ca2+ -CAM的自由浓度如何受到自由Ca2+浓度和神经元素表达的局部升高的影响，以及这与活动的变化如何相关，这与表达的CAM依赖性环化酶活性（AC1）如何相关。我们还将研究消除其CAM水槽功能的神经统治的PKC-催化的磷酸化是否可以动态控制游离CA2+CAM浓度和环化酶活性。鉴于细胞中CAM的限制量，了解它是如何在CAM依赖性酶之间分布的，以及这如何影响细胞功能，显然至关重要。该提议强调了在质膜处增强自由Ca2+cam浓度的因素，并控制CAM依赖性循环酶以及其他低亲和力CAM依赖性酶的活性。这些循环酶是一类特别重要的低亲和力CAM靶标，已经特别与腮腺腺泡和肾上腺肾小球细胞的分泌和类固醇生成有关，以及某些形式的学习形式。