title: A high-capacity membrane potential FRET-based assay for the sodium-coupled glucose co-transporter SGLT1.
abstract: Secondary active glucose transport is mediated by at least four members of the solute-linked carrier 5 gene family (sodium/glucose transporter [SGLT] 1-4). Human genetic disorders of SGLTs including glucose-galactose malabsorption and familial renal glucosuria have increased attention on members of this family of transporters as putative drug targets. Using human SGLT1 (hSGLT1) as a paradigm, we developed a functional assay that should be adaptable to ultra-high-throughput operation and to other SGLTs. Human embryonic kidney (HEK) 293 cells stably expressing hSGLT1 (hSGLT1/HEK293 cells) display a Na(+)-dependent, phlorizin-sensitive alpha-methyl-D-[(14)C]glucopyranoside flux with expected kinetic parameters. In electrophysiological studies with hSGLT1/HEK293 cells, substrate-dependent changes in membrane potential were observed, consistent with the electrogenic operation of hSGLT1. With the use of voltage-sensitive dyes, a membrane potential, fluorescence resonance energy transfer-based functional assay on a voltage/ion probe reader platform has been established for SGLT1. This high-capacity functional assay displays similar characteristics in terms of substrate specificity and phlorizin sensitivity to those determined by more traditional approaches and should provide a means to identify novel and selective SGLT inhibitors.
authors.affiliation: Department of Ion Channels, Merck Research Laboratories, Rahway, NJ 07065, USA. adam_weinglass@merck.com
authors.forename: Adam B
authors.initials: AB
authors.lastname: Weinglass
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Swensen
authors.forename: Jessica
authors.initials: J
authors.lastname: Liu
authors.forename: William
authors.initials: W
authors.lastname: Schmalhofer
authors.forename: Anu
authors.initials: A
authors.lastname: Thomas
authors.forename: Brande
authors.initials: B
authors.lastname: Williams
authors.forename: Leila
authors.initials: L
authors.lastname: Ross
authors.forename: Kristina
authors.initials: K
authors.lastname: Hashizume
authors.forename: Martin
authors.initials: M
authors.lastname: Kohler
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kaczorowski
authors.forename: Maria L
authors.initials: ML
authors.lastname: Garcia
mesh_terms: D001463:Barbiturates; D002460:Cell Line; D004396:Coloring Agents; D003374:Coumarins; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004594:Electrophysiology; D004983:Ethanolamines; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007555:Isoxazoles; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010695:Phlorhizin / Q000494:pharmacology; D051269:Sodium-Glucose Transporter 1 / Q000032:analysis* / Q000378:metabolism; D013858:Thiobarbiturates
chemical_list: D001463:Barbiturates; D004396:Coloring Agents; D003374:Coumarins; D004983:Ethanolamines; D007555:Isoxazoles; C502247:N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D013858:Thiobarbiturates; C025284:bis(1,3-diethylthiobarbiturate)trimethineoxonol; D010695:Phlorhizin