Neutralization of a conserved amino acid residue in the human Na+/glucose transporter (hSGLT1) generates a glucose-gated H+ channel.
The role of conserved Asp204 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 microm) 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.
Quick
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Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1751, USA. mquick@mednet.ucla.edu
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D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D011522:Protons; D051269:Sodium-Glucose Transporter 1
D017931:DNA Primers; D007473:Ion Channels; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D005947:Glucose