Self-referencing, non-invasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux

Biological systems have very different internal ion compositions in compari son with their surrounding media. The difference is maintained by transport mechanisms across the plasma membrane and by internal stores. On the plasm a membrane, we can classify these mechanisms into three types, pumps, porte rs, and channels. Channels have been extensively studied, particularly sinc e the advent of the patch clamp technique, which opened new windows into io n channel selectivity and dynamics. Pumps, particularly the plasma membrane Ca2+-ATPase, and porters are more illusive. The technique described in thi s paper-the self-referencing, ion-selective (or Seris) probe, has the abili ty to monitor the behavior of membrane transport mechanisms, such as the pu mps and porters, in near to real-time by non-invasively measuring local ext racellular ion gradients with high sensitivity and square micron spatial re solution. The principles behind the self-referencing technique are described with an overview of systems utilizing ion, electrochemical and voltage sensors. Eac h of these sensors employs the simple expedient of increasing the system re solution by self-referencing and, thereby, removing the drift component inh erent to all electrodes. The approach is described in detail, as is the man ner in which differential voltage measurements can be converted into a flux value. For the calcium selective probes, we can resolve flux values in the low to sub pmol.cm(-2)s(-1) range. Complications in the use of the liquid ion exchange cocktail are discussed. Applications of the calcium selective probe are given, drawing on examples from the plant sciences,developmental biology, muscle physiology, and the neurosciences. (C) 1999 Wiley-Liss, Inc .