Study of Reconstitution of the Rabbit Parotid Na⁺/K⁺/2Cl^-Cotransporter

Abstract

The critical role of Cl^- transepithelial transport in eliciting a normal fluid secretion or absorption in various epithelia is strongly suggested from clinical and pathological studies. Modifications of epithelial function caused from bacterial toxins or genetically determined, as in cystic fibrosis, appear to be specifically dependent on some alteration in Cl^- transport across epithelia. The apical and basolateral membranes of epithelial cells are endowed with Cl^- transport systems of different specificities and characteristics. According to the current view on ion transepithelial transport, the entry of Cl^- into the cell can occur via cation coupled transport systems. In many absorbing and secreting epithelia a quaternary cotransport system of stoichiometry lNa⁺: 1K⁺:2C1^- has been characterized. In order to understand the mechanism of transport and ion coupling of such complex transport system, it is important to have it in a functional state in reconstituted systems. In this type of study the availability of an high affinity inhibitor of the transporter can provide a useful tool to label the protein in various steps of solubilization and reconstitution. As regards the Na⁺/K⁺/2Cl^- cotransporter the appropriate labelling agent to use for this purpose appears to be bumetanide.