MAGNESIUM REGULATION OF MEMBRANE FUNCTION IN PARAMECIUM

镁是许多细胞内过程的重要物质。大多数单元格 含有维持细胞质内游离镁离子的特殊运输系统 浓度([Mg~(2+)]i)在确保最佳活性的水平。然而， 最近的证据表明，荷尔蒙导致某些细胞内[镁离子]i的变化 细胞，这意味着这种阳离子被用来调节细胞功能。在……里面 此外，各种疾病状态-包括糖尿病和 高血压-与异常的镁离子水平有关，这表明 镁离子动态平衡缺陷可能与其发病有关。这些 观察是深刻和深远的，然而，尽管潜在的 镁离子的重要性对于[镁离子]i是如何存在的知之甚少 或[Mg~(2+)]i如何调控细胞活动。这部分是因为 因为[Mg2+]i在相对较少的(通常是非 可兴奋)细胞，而且因为[Mg2+]i的变化经常被证明是 小而慢，妨碍了对其后果的分析。这项建议 以草履虫为研究对象，研究了[Mg2+]i在真核生物中的功能 一个模范系统。使用草履虫有许多令人信服的原因， 包括增长要求的简单性，这是大多数 微生物，易于基因突变和操纵，表现为 膜电活动在细胞的游泳行为中的作用。主 关注草履虫的原因在于这个单细胞 表达一个大的快速激活的内向镁离子电流(i-mg) 使用传统的电压钳位，电流易于触发和监控 技术，从而提供了一个真正无与伦比的机会来研究 [Mg~(2+)]i变化对细胞活性的影响和作用。这 提案有三个具体目标：(A)描述特定于镁离子的通道 这被认为是导致镁离子内流的原因，(B)使用电压- 以及膜片钳技术，以确定这种镁离子内流是如何修改的 膜兴奋性(具体地说，通过抑制K通道和作用 作为激活依赖于镁离子的氯离子通道的第二信使)，以及 (C)启动对i-mg及其调控途径的遗传解剖，以及 镁离子内流的靶标。确定其作用和分子组成 草履虫中的镁离子动态平衡将对 这种阳离子在高等真核生物中的功能，通过揭示 先前未知的镁离子调节通路，很可能有助于揭示 高血压的成因和发展。

Magnesium is a vital for numerous intracellular processes. Most cells contain specialized transport systems that maintain cytoplasmic free Mg2+ concentrations ([Mg2+]i) at levels that ensure optimal activity. However, recent evidence suggests that hormones cause [Mg2+]i to change in some cells, implying that this cation is used to regulate cell function. In addition, a variety of diseased states - including diabetes and hypertension - have been linked with abnormal Mg2+ levels, suggesting that defects in Mg2+ homeostasis may be involved in their etiology. These observations are profound and far-reaching, yet despite the potential importance of Mg2+ relatively little is known about how [Mg2+]i is regulated or how [Mg2+]i might regulate cell activity. This is partly because [Mg2+]i has been monitored in relatively few (and often non- excitable) cells, and because changes in [Mg2+]i have often proved to be small and slow, hampering analysis of their consequences. This proposal investigates the functions of [Mg2+]i in eukaryotes, using Paramecium as a model system. There are many compelling reasons for using Paramecium, including a simplicity of growth requirements that is typical of most microbes, ease of genetic mutation and manipulation, the manifestation of membrane electrical activity in the cell's swimming behavior. The main reason for focussing on Paramecium lies in the fact that this unicell expresses a large and fast-activating, inward Mg2+ current (I-Mg) The current is easy to trigger and monitor using conventional voltage-clamp techniques, thus providing a truly unparalleled opportunity to examine the consequences and role of changes in [Mg2+]i for cell activity. This proposal has three specific aims: (A) characterize a Mg2+-specific channel that is believed to be responsible for the Mg2+ influx, (B) use voltage- and patch-clamp techniques to define how this Mg2+ influx modifies membrane excitability (specifically, by inhibiting K channels and acting as a second messenger for activation of a Mg2+-dependent Cl channel), and (C) initiate a genetic dissection of I-Mg, its regulatory pathways, and the targets of Mg2+ influx. Defining the role and molecular components of Mg2+ homeostasis in Paramecium will produce invaluable insights into the functions of this cation in higher eukaryotes and, by revealing previously-unsuspected Mg2+-regulated pathways, may well shed light on the causes and development of hypertension.