Inhibitor binding in the human renal low- and high-affinity Na+/glucose cotransporters

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'-beta-glucoside). In this study, we examined the inhibition of alpha-methyl-d-glucopyranose transport by phlorizin and a new candidate drug, sergliflozin-A [(2-[4-methoxyphenyl]methyl)phenyl beta-d-glucopyranoside], in COS-7 cells expressing hSGLT1 and hSGLT2. Inhibition by phlorizin was competitive, with K(i) values of 0.3 muM in hSGLT1 and 39 nM in hSGLT2. Inhibition by sergliflozin-A was also competitive, with K(i) values of 1 muM 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 IC(50) values of 142 muM in hSGLT1 and 25 muM 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 IC(50) for all inhibitors. In contrast, mutagenesis of the analogous Cys615 in hSGLT2 produced the opposite effect, a decrease in IC(50) 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.