COMBINED WHOLE-CELL AND SINGLE-CHANNEL CURRENT MEASUREMENT WITH QUANTITATIVE CA2+ INJECTION OR FURA-2 MEASUREMENT OF CA2-

Publisher Summary Intracellular free Ca 2+ is an important second messenger in most cell types and cytoplasmic Ca 2+ concentration, [Ca 2+ ] i , is tightly controlled by cytoplasmic buffering and regulation of influx and efflux pathways. In excitable cells, cytoplasmic Ca 2+ couples electrical events with a broad range of effector functions. Voltage-dependent calcium channels provide a direct means for this coupling, while postsynaptic ligand-gated channels provide additional regulated pathways for Ca 2+ entry. Techniques for introducing Ca 2+ into the cytoplasm are well established and several of these methods have been refined so that reasonably quantitative introductions of Ca 2+ can be made. Recent years have seen rapid advances in techniques for measuring [Ca 2+ ] i using fluorescent dyes, but older methods, utilizing aequorin, arsenazo, and ionsensitive microelectrodes, take these measurements back several decades. This chapter gives an example in which these three techniques are combined in an experiment in a single cell. The [Ca 2+ ] i is changed in a quantitative manner as the result of controlled influx through voltage-gated calcium channels. The change in [Ca 2+ ] i is measured using fluorescent imaging with the Ca 2+ -sensitive dye Fura-2 (Molecular Probes, Eugene, OR), and membrane (tail) currents are measured. The strength of combining these techniques is apparent in this experiment where it was possible lo control [Ca 2+ ] i quantitatively and observe its effects on the activation of Ca 2+ - activated currents. Experiments combining measurements of membrane currents and [Ca 2+ ] i with the controlled introduction of Ca 2+ into the cytoplasm have provided important insight into cellular Ca 2+ regulation and signaling. This chapter discusses various combinations of these techniques that allow measurement or control of [Ca 2+ ] i and simultaneously allow the measurement of membrane currents. These combinations, while adding to the complexity of the experiment, thereby sometimes limiting the available preparations, are necessary in order to study control of [Ca 2+ ] i and its role in such important neuronal processes as excitation-gating coupling.