I propose to study the regulation of intracellular Ca2+ ([Ca]i) in the colon carcinoma cell line HT-29/B6, which has been introduced as a C1- and mucus-secreting epithelium. This issue is of great importance, because [Ca]i has been shown to fulfill a regulatory role during secretory events in intestinal cells and in many other epithelial cell types. First, I will characterize the plasma membrane Ca2+ permeability in HT-29/B6 using as a measure for Ca2+ entry the concentration of [Ca]i as determined by means of fura-2 fluorescence, as well as the amount of quenching of the fura-2 signal by Mn2+. Once the data have been obtained with isolated cells, the experiments will be repeated on intact epithelial sheets to assure that measurements in the single cells are physiological. A second set of experiments will aim to identify the ionic channels) underlying the plasma membrane Ca2+ conductance using fluctuation analysis and patch-clamp. The basic idea here is to apply fluctuation analysis on macroscopic Ca2+ currents through apically permeabilized sheets and mediated by the basolateral Ca2+ channels. An alternative approach will be to record whole-cell patch-clamp Ca2+ currents and their fluctuation in intact isolated cells. The whole-cell patch-clamp and fluctuation analysis configuration will also be used in conjunction with the newly developed mag-fura 2 fluorescence methodology to investigate the relationship between the Ca2+ content of the intracellular Ca2+ stores and Ca2+ entry through the plasma membrane Ca2+ channels, which is the third major aim of this research proposal. Summarizing, the combination of advanced optical and electrophysiological techniques proposed in this project will allow me to determine the properties of and the interactions between the plasma membrane Ca2+ channels and the internal store, thereby contributing to a more complete picture of Ca2+ signalling in an intact cell.
