CALMODULIN FUNCTION IN THE CELL

细胞中的钙调蛋白功能

• 批准号: 2908126
• 负责人: ANTHONY J PERSECHINI
• 金额: $ 25.82万
• 依托单位: UNIVERSITY OF ROCHESTER
• 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 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2908126
• 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.

当钙调蛋白 (CaM) 与 Ca2+ 结合时，它会激活大量在细胞中具有关键功能的不同酶。我们的初步结果表明细胞中 Ca2+-CaM 的最大整体游离浓度约为 30 nM，并且 CaM 的总浓度远低于 CaM 结合蛋白的浓度。虽然许多 CaM 依赖性酶以大约 1 nM 的解离常数结合 Ca2+-CaM，但有些酶的亲和力低数百倍。此类酶包括 CaM 依赖性腺苷酸环化酶，可被细胞中的 Ca2+ 强力激活。这与我们测量的 Ca2+-CaM 的整体低游离浓度不一致。然而，在几种细胞类型中，似乎需要在质膜上产生游离 Ca2+ 浓度局部增加的条件来显着激活环化酶。它们与神经调节蛋白或神经颗粒蛋白共同定位在大脑中，这些膜相关蛋白被认为具有 CaM“下沉”的功能。因此，我们假设存在两种机制，可以增强质膜上的游离 Ca2+-CaM 浓度，从而产生观察到的环化酶激活：(1) 由于 Ca2+ 局部升高而扩散招募 CaM，以及 (2) CaM 位于质膜上，由 CaM 汇蛋白形成。我们建议通过确定质膜上产生的 Ca2+-CaM 的游离浓度如何受到游离 Ca2+ 浓度和神经调节蛋白表达的局部升高的影响，以及这与表达的 CaM 依赖的活性变化有何关系来检验这一假设。环化酶活性（AC1）。我们还将研究 PKC 催化的神经调节蛋白磷酸化（消除其 CaM 库功能）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 的数量有限，了解它如何在 CaM 依赖性酶之间分布以及这如何影响细胞功能显然具有普遍重要意义。该提案强调了提高质膜上游离 Ca2+-CaM 浓度并控制 CaM 依赖性环化酶以及其他低亲和力 CaM 依赖性酶活性的因素。环化酶是一类特别重要的低亲和力 CaM 靶标，与腮腺腺泡和肾上腺球细胞的分泌和类固醇生成以及某些形式的学习有关。