Transport of lactate: Characterization of the transporters involved in transport at the plasma membrane by heterologous protein expression in Xenopus oocytes

Abstract

Lactate transport in the brain and other organs is mediated by the monocarboxylate transporters MCT1-4. To study the transporters involved in lactate fl ux, various techniques such as in vivo measurements of lactate concentrations by magnetic resonance imaging, determination of lactate-induced proton fl uxes in brain slices and cell cultures using fl uorescent dyes and pH-sensitive microelectrodes, or uptake studies of radioactively labeled lactate in cell cultures have been applied. However, characterization of distinct MCT isoforms in brain slices and cell cultures can be diffi cult, since different isoforms of the transporter are found in a single cell type and transport activity can be infl uenced by other proteins. A powerful tool for the characterization of transporters such as the MCTs is heterologous protein expression in Xenopus laevis oocytes. The expression system makes it easy to introduce mutations in the transporter and allows co- expression of MCTs with other proteins to study mechanisms, regulation, and modulation of these lactate transporters. In this chapter the characterization of MCTs, heterologously expressed in Xenopus oocytes, by use of pH-sensitive microelectrodes is described. Detailed information on the preparation of Xenopus oocytes and the injection of cRNA is given. Furthermore, a protocol for the production and usage of both singleand double-barreled pH-sensitive microelectrodes is provided. In addition, analysis of the measurements, as well as calculation of buffer capacities and MCT-mediated substrate fl uxes is discussed.