Haas M, McManus T J
Methods Enzymol. 1989;173:280-91. doi: 10.1016/s0076-6879(89)73018-4.
Two methods have been described for the study of cation-chloride cotransport systems. The zero-trans efflux method is designed to determine stoichiometric relationships between cotransported ions under conditions where ion exchanges cannot occur. These exchanges (e.g., Na+/Na+, K+/K+) may occur as partial or incomplete reactions of a cotransport process and can lead to erroneous determinations of the stoichiometry of the cotransport process. The zero-trans efflux method can also be used to study the effects of cell volume, pH, and intracellular ion concentrations on cotransport processes. The valinomycin method is used to determine the electrogenicity or electroneutrality of transport, and in this regard can be used in conjunction with other methods such as those employing potential-sensitive dyes or microelectrodes. Other, more recently developed ionophores with specificity for lithium rather than potassium have now been used to study the effect of Em on the ATP-dependent Na+/K+ pump. It may be possible to use such ionophores to confirm the suspected electroneutrality of (K+ + Cl-) cotransport systems, as well as for other studies of specific potassium transport processes in which valinomycin obviously cannot be used. Both methods discussed in detail in this chapter, and particularly the valinomycin method, were originally devised for use in red blood cells in order to take advantage of (or circumvent) properties of the red cell membrane, such as its low permeability to sodium and potassium and relatively high permeability to chloride. However, valinomycin has been used successfully to demonstrate the electroneutrality of (Na+ + K+ + 2Cl-) cotransport in MDCK cells, and the zero-trans efflux method should be applicable to the study of transport processes in other types of cells in suspension, so long as the transport system being studied can be accurately defined (e.g., as an inhibitor-sensitive or chloride-dependent cation flux) and comprises a significant fraction of the total salt efflux.
