38] Fluorescence techniques for studying cloned channels and transporters expressed in Xenopus oocytes

Abstract

This chapter describes some applications of fluorescence to the study of transporters and channels expressed in Xenopus laevis oocytes. The first approach involves the measurement of changes in surface fluorescence associated with conformational transitions or gating of exogenously expressed channels and transporters. This approach is based on changes in the fluorescence of covalently incorporated probes resulting from perturbation of the probe microenvironment during a process such as channel opening. The second approach involves the use of ion-sensitive fluorescent dyes to analyze solute movements during transport. The methods for combining each technique with measurement of membrane currents under voltage clamp are described in the chapter. Valuable information about conformational changes in transmembrane proteins can be obtained from Xenopus oocytes in situ with site-directed fluorescent labeling. After using site-directed mutagenesis to substitute cysteine for a particular residue in extracellularly exposed regions of the protein, an extrinsic sulfhydryl-reactive fluorescent probe can be covalently attached to this residue. By using a fluorescent probe whose quantum yield is highly dependent on its environment, changes in the environment of the labeled residue resulting from conformational changes in the protein can be monitored via changes in the fluorescence intensity and spectrum of the probe from the surface of Xenopus oocytes.