title: The Oscillating Stimulus Transporter Assay, OSTA: Quantitative Functional Imaging of Transporter Protein Activity in Time and Frequency Domains.
abstract: 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 presented in which oscillating stimuli are presented to transporter-expressing cells, and activity is measured through imaging the corresponding oscillating responses of intracellular fluorescent sensors. This approach yields continuous temporal readouts of transporter activity and can therefore be used to measure time-dependent responses to drugs and other stimuli. Because of the periodic nature of the response, temporal Fourier transforms can be used to identify and quantify regions of interest in the xy plane and to overcome noise. 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.
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA. Electronic address: jacobpkeller@gmail.com.
authors.forename: Jacob P
authors.initials: JP
authors.lastname: Keller
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA. Electronic address: loogerl@janelia.hhmi.org.
authors.forename: Loren L
authors.initials: LL
authors.lastname: Looger
mesh_terms: D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism; D001559:Benzhydryl Compounds / Q000494:pharmacology; D001578:Benzopyrans / Q000737:chemistry*; D001681:Biological Assay; D001692:Biological Transport; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D027963:Chloride-Bicarbonate Antiporters / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D027342:Excitatory Amino Acid Transporter 2; D005456:Fluorescent Dyes / Q000737:chemistry*; D051273:Glucose Transporter Type 2 / Q000235:genetics / Q000378:metabolism; D005960:Glucosides / Q000494:pharmacology; D050491:Glutamate Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D061848:Optical Imaging / Q000379:methods*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D020156:Salicylic Acid / Q000494:pharmacology; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism; D051297:Sodium-Glucose Transporter 2 / Q000235:genetics / Q000378:metabolism; D000076988:Sulfate Transporters; D014162:Transfection
chemical_list: D027321:Anion Transport Proteins; D001559:Benzhydryl Compounds; D001578:Benzopyrans; D027963:Chloride-Bicarbonate Antiporters; D027342:Excitatory Amino Acid Transporter 2; D005456:Fluorescent Dyes; D051273:Glucose Transporter Type 2; D005960:Glucosides; D050491:Glutamate Plasma Membrane Transport Proteins; C493397:SLC1A2 protein, human; C089651:SLC26A2 protein, human; C081011:SLC26A3 protein, human; C495680:SLC2A2 protein, human; C498898:SLC4A4 protein, human; C089180:SLC5A2 protein, human; C063917:SNARF dye; D027982:Sodium-Bicarbonate Symporters; D051297:Sodium-Glucose Transporter 2; D000076988:Sulfate Transporters; C529054:dapagliflozin; D020156:Salicylic Acid
keywords: Fourier transform; biosensor; drug screening; fluorescence imaging; functional assay; functional imaging; high throughput; inhibitor; transporter