Topology of the Shaker potassium channel probed with hydrophilic epitope insertions

Abstract

The structure of the Shaker potassium channel has been modeled as passing through the cellular membrane eight times with both the NH2 and COOH termini on the cytoplasmic side (Durrell, S.R., and H.R. Guy. 1992. Biophys. J. 62:238-250). To test the validity of this model, we have inserted an epitope consisting of eight hydrophilic amine acids (DYKDDDDK) in predicted extracellular and intracellular loops throughout the channel. The channels containing the synthetic epitope were expressed in Xenopus oocytes, and function was examined by two-electrode voltage clamping. All of the mutants containing insertions in putative extracellular regions and the NH2 and COOH termini expressed functional channels, and most of their electrophysiological properties were similar to those of the wild-type channel. Immunofluorescent staining with a monoclonal antibody against the epitope was used to determine the membrane localization of the insert in the channels. The data confirm and constrain the model for the transmembrane topology of the voltage-gated potassium channel.