Anion Transport Systems: Continuous Monitoring of Transport by Fluorescence (CMTF) in Cells and Vesicles

Abstract

Anion transport mechanisms are found in mammalian cell membranes in the form of exchangers or antiporters (Passow, 1987; Aronson, 1989), cation-anion cotransporters or symporters (Hoffman, 1986; O’Grady et al., 1987) and Cl channels (Greger, 1985, 1988; Gogelein, 1988; Frizzell et al., 1986). The transport of the anions across both biological and artificial membranes are studied by following the transcompartmental movement of physiologically relevant substrates such as Cl⁻, HCO₃, SO4⁻² or H₂PO₄ ⁻. For electroneutral mechanisms, the anion flux can be traced by a variety of physical or chemical techniques, the most widely used being tracing the movement of a radiolabeled anion by separating the compartments at various time points, sampling their contents, and measuring the amount of tracer in one of them. This method is, however, limited by the space available to the substrate, its specific activity and total radioactivity, the efficiency and speed of separation relative to the actual transport rates, as well as by the capacity of the biological system to retain its structural integrity during separation steps. Since this method is discrete in nature, the amount of information it can yield is usually limited and often not sufficiently precise for a thorough kinetic evaluation. For charge-conducting transport systems, the classical and most straightforward approach is the electrophysiological one, which is used in various forms according to the level of information required and the nature of the biological system. Electrophysiological techniques are of an invasive nature and most commonly inaccessible to small organelles. On the other hand for conductive systems operating in cells and vesicles, isotopic techniques can still be very useful when used under conditions where a large chemical gradient of the ion is established across the membrane and a radio-labeled tracer of the ion is placed at the low concentration side (Garty et al., 1983). In such conditions, the diffusion potential created across the membrane by the transportable ion will drive the isotopic species into the vesicles at a rate commensurate with the potential and the intrinsic conductivity of the channel tested (Landry et al., 1987; Breuer, 1989).

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