MAGNESIUM REGULATION OF MEMBRANE FUNCTION IN PARAMECIUM

镁对许多细胞内过程至关重要。大多数细胞 含有专门的运输系统，维持细胞质游离Mg2 + 浓度（[Mg2 +] i）在确保最佳活性的水平。但是，在这方面， 最近的证据表明，激素导致[Mg2 +] i在某些情况下发生变化， 细胞，这意味着这种阳离子是用来调节细胞功能。在 此外，各种疾病状态-包括糖尿病和 高血压-与异常的Mg2+水平有关，这表明， Mg 2+稳态的缺陷可能参与其病因学。这些 观察是深刻和深远的，但尽管有潜力， Mg2+的重要性相对较少了解[Mg2 +] i是如何 调节或[Mg2 +] i如何调节细胞活性。这部分是 因为[Mg2 +] i已经在相对少的（并且通常非- 可兴奋的）细胞，并且因为[Mg2 +] i的变化经常被证明是 小而慢，阻碍了对其后果的分析。这项建议 以草履虫为材料，研究了[Mg 2 +] i在真核生物中的功能。 一个模型系统使用草履虫有许多令人信服的理由， 其中包括大多数公司通常要求的简单的增长要求， 微生物，基因突变和操纵的容易性， 细胞游泳行为中的膜电活动。主要 关注草履虫的原因在于， 表达一个大的和快速激活的，向内的Mg 2+电流（I-Mg）。 使用传统的电压箝位电路，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，检查 [Mg2 +] i变化对细胞活性的影响和作用。这 该提案具有三个具体目标：（A）表征Mg 2+特异性通道 这被认为是负责Mg 2+流入，（B）使用电压- 和膜片钳技术来确定这种Mg 2+流入如何改变 膜兴奋性（特别是，通过抑制K通道和作用 作为激活Mg2+依赖性Cl通道的第二信使），和 (C)启动I-Mg的遗传解剖，其调节途径， Mg2+内流的靶点。定义的作用和分子组成 草履虫中的Mg 2+稳态将产生对 这种阳离子在高等真核生物中的功能，并通过揭示 以前未被怀疑的Mg 2+调节途径，很可能揭示 高血压的病因和发展。

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.