DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

点击翻译按钮获取中文摘要

The development of intracellular indicators for cytosolic cations and other parameters of interest has had a major impact on the field of cell biology. The concentrations of cellular cations play fundamental roles in the mediation of hormonal signaling, and are also involved in the mediation of cell injury. One of the major achievements of our group has been the development of the APTRA series of chelators, which are widely used for the assessment of intracellular Mg2+ concentration. Despite the utility of APTRA and its fluorescent and fluorinated derivatives for the determination of intracellular Mg2+ levels, it would be desirable to develop and indicator with greater Mg2+/Ca2+ selectivity. During the past year, we made major progress in this direction. A series of indicators with very promising characteristics is currently being synthesized and will be evaluated further in the near future. One of the challenging aspects of the use of indicators for determining the levels of intracellular ions is the problem of intracellular organelles. Depending on the loading of the indicators into different cellular compartments, the information derived can be very misleading. Although the ability of fluorescent indicators to handle such problems is inherently limited, NMR indicators provide a much better opportunity for the assessment of subcellular ion concentrations. In collaboration with Drs. Elizabeth Murphy and Weina Chen, we have found that the recently developed fluorinated calcium indicator TFBAPTA, provides a measure of both cytosolic and sarcoplasmic reticulum calcium pools. These are the first direct measurements of SR calcium ion concentrations. Further studies of the effects of various chemical agents on the SR Ca2+ pool are in progress. The discovery by our group that fluorinated compounds give distinct chemical shifts in an intracellular environment has allowed studies of the distribution and transport rates of various fluorodeoxyglucose analogs provide detailed insight into the transport process.