Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters

• Published in: British journal of pharmacology Vol. 134; no. 3; pp. 484 - 495
• Main Authors: Hirayama, Bruce A, Díez‐Sampedro, Ana, Wright, Ernest M
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.10.2001; Nature Publishing
• Subjects: Animals; Biological and medical sciences; Carrier Proteins - antagonists & inhibitors; Carrier Proteins - biosynthesis; Carrier Proteins - physiology; Cell physiology; electrophysiology; Esculin - chemistry; Esculin - pharmacokinetics; Female; Fundamental and applied biological sciences. Psychology; GABA; GABA Plasma Membrane Transport Proteins; General pharmacology; glucose; Humans; inhibitor binding; Medical sciences; Membrane and intracellular transports; Membrane Glycoproteins - antagonists & inhibitors; Membrane Glycoproteins - biosynthesis; Membrane Glycoproteins - physiology; Membrane Potentials - drug effects; Membrane Potentials - physiology; Membrane Proteins - antagonists & inhibitors; Membrane Proteins - biosynthesis; Membrane Proteins - physiology; Membrane Transport Proteins; Molecular and cellular biology; Monosaccharide Transport Proteins - antagonists & inhibitors; Monosaccharide Transport Proteins - biosynthesis; Monosaccharide Transport Proteins - physiology; Organic Anion Transporters; Pharmacokinetics. Pharmacogenetics. Drug-receptor interactions; Pharmacology. Drug treatments; pharmacophore; Phlorhizin - chemistry; Phlorhizin - pharmacokinetics; sodium; Sodium substrate cotransport; Sodium-Glucose Transporter 1; Xenopus laevis; Human origin; Biological transport; Amphibia; Electrophysiology; Binding site; Glucose; Hydrophobicity; Absorption; Structure activity relation; Baclofen; Recombinant protein; Human; Drug; Xenopus laevis; Salientia; Prodrug; In vitro; Vertebrata; Sodium; Distribution; Excitatory aminoacid; GABA; Neurotransmitter; Inhibitor; Models; Kinetics; Oocyte
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0704274

Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate‐dependent current and inhibitor‐induced changes in cotransporter conformation. The competitive, non‐transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5‐orders of magnitude. Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7×12Å. Important hydrogen bond interactions occur at five positions bordering this surface. In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site ∼8Å from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non‐competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1). Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non‐radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors. British Journal of Pharmacology (2001) 134, 484–495; doi:10.1038/sj.bjp.0704274