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.
