The New “Patch Clamping” for Transporters: Oscillating Perfusion and Quantitative Functional Imaging of Biosensors for Transporter Functional Measurements

Transmembrane transporter proteins allow the passage of essentially all biologically important molecules across the lipid membranes of cells and organelles and are therefore of central importance to all forms of life. Current methods of transporter measurement, however, are lacking in several dimensions. Herein, a method is described in which oscillating perfusion stimuli are presented to transporter-expressing cells or tissues, and activity is measured through imaging the corresponding oscillating fluorescence responses of intracellular fluorescent sensors. This approach yields continuous temporal readouts of transporter flux and can therefore be used to discover and measure time-dependent responses to drugs and other stimuli. This technique, called the Oscillating Stimulus Transporter Assay (OSTA), should greatly facilitate both functional characterization of transporters as well as high-throughput screening of drugs for transporters of particular pathophysiological interest. As such, OSTA may do for transporters what patch clamping did for ion channels