TY - JOUR T1 - Inhibitor Binding in the Human Renal Low- and High-Affinity Na<sup>+</sup>/Glucose Cotransporters JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 985 LP - 991 DO - 10.1124/jpet.107.129825 VL - 324 IS - 3 AU - Ana M. Pajor AU - Kathleen M. Randolph AU - Sandy A. Kerner AU - Chari D. Smith Y1 - 2008/03/01 UR - http://jpet.aspetjournals.org/content/324/3/985.abstract N2 - The kidney contains two Na+/glucose cotransporters, called SGLT2 and SGLT1, arranged in series along the length of the proximal tubule. The low-affinity transporter, SGLT2, is responsible for the reabsorption of most of the glucose in the kidney. There is recent interest in SGLT2 as a target for the treatment of type II diabetes using selective inhibitors based on the structure of the phenylglucoside, phlorizin (phloretin-2′-β-glucoside). In this study, we examined the inhibition of α-methyl-d-glucopyranose transport by phlorizin and a new candidate drug, sergliflozin-A [(2-[4-methoxyphenyl]methyl)phenyl β-d-glucopyranoside], in COS-7 cells expressing hSGLT1 and hSGLT2. Inhibition by phlorizin was competitive, with Ki values of 0.3 μM in hSGLT1 and 39 nM in hSGLT2. Inhibition by sergliflozin-A was also competitive, with Ki values of 1 μM in hSGLT1 and 20 nM in hSGLT2. Phloretin [3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone; the aglucone of phlorizin] was a less potent inhibitor, with IC50 values of 142 μM in hSGLT1 and 25 μM in hSGLT2. Site-directed mutagenesis of residues believed to be in the phlorizin binding site showed that only Cys610 is involved in inhibitor binding in the human transporters. Mutation of Cys610 in hSGLT1 to lysine resulted in an increased IC50 for all inhibitors. In contrast, mutagenesis of the analogous Cys615 in hSGLT2 produced the opposite effect, a decrease in IC50 for phlorizin and sergliflozin-A. The differences in the effects of the mutations between hSGLT1 and hSGLT2 suggest that this cysteine holds key residues in place rather than participating directly in inhibitor binding. The American Society for Pharmacology and Experimental Therapeutics ER -