Functional Asymmetry of the Human Na+/Glucose Transporter (hSGLT1) in Bacterial Membrane Vesicles

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Abstract
The functional characteristics of the forward and reverse transport modes of the human Na+/glucose transporter (hSGLT1) were investigated using plasma membrane vesicles of E. coli expressing the recombinant transporter. Correctly and inverse-oriented vesicles were employed to measure the initial rates of methyl-α-d-glucose uptake, under zero-trans conditions, as a function of Na+, sugar, and phlorizin concentrations and membrane potential. This approach enabled the analysis of the two faces of hSGLT1 in parallel, revealing the reversibility of Na+/sugar cotransport. While the key characteristics of secondary active sugar transport were maintained in both modes, namely, Na+ and voltage dependence, the kinetic properties of the two sides indicated a functional asymmetry of the transporter. That is, the apparent affinity for sugar and driver cation Na+ exhibited a difference of more than 1 order of magnitude between the two modes. Furthermore, the selectivity pattern of ligands and the interaction of the transporter with the competitive inhibitor phlorizin were different. Whereas the high-affinity substrates, d-glucose and d-galactose, inhibited uptake of radioactive sugar tracer at their physiological concentrations (10 mM) in the forward reaction, they were poor inhibitors even at high concentrations in the reverse transport mode. Taken together, these results confirm the successful employment of E. coli to express and characterize a human membrane protein (hSGLT1), elucidating the functional asymmetry of this cotransporter.