Neutralization of a conserved amino acid residue in the human Na+/glucose transporter (hSGLT1) generates a glucose-gated H+ channel

The role of conserved Asp(204) in the human high affinity Na+/glucose cotransporter (hSGLT1) was investigated by site-directed mutagenesis combined with functional assays exploiting the Xenopus oocyte expression system. Substitution of H+ for Na+ reduces the apparent affinity of hSGLT1 for glucose from 0.3 to 6 mM. The apparent affinity for H+ (7 muM) is about three orders of magnitude higher than for Na+ (6 mM). Cation/glucose cotransport exhibits a coupling ratio of 2 Na+ (or 2 H+):1. Pre-steady-state kinetics indicate that similar Na+- or H+-induced conformational changes are the basis for coupled transport. Replacing Asp204 with Glu increases the apparent affinity for H+ by >20-fold with little impact on the apparent Na+ affinity. This implies that the length of the carboxylate side chain is critical for cation selectivity. Neutralization of (Asp204 Asp --> Asn or Cys) reveals glucose evoked H+ currents that were one order of magnitude greater than Na+ currents. These phlorizin-sensitive H+ currents reverse and are enhanced by internal acidification of oocytes. Together with a H+ to sugar stoichiometry as high as 145:1, these results favor a glucose-gated H+ channel activity of the mutant. Our observations support the idea that cotransporters and channels share common features.