SODIUM D-GLUCOSE COTRANSPORTER CHARGE MOVEMENTS INVOLVE POLAR RESIDUES

Na+-dependent glucose transporters (SGLT1) exhibit transient carrier c urrents with a time constant (tau) of 2-20 ms, and the charge transfer (Q) fits the Boltzmann equation. There is a 60-mV negative displaceme nt in the tau/V and Q/V curves between the human and rabbit SGLT1 prot eins, and the initial goal was to identify the charges responsible for these differences in kinetics. We have focused on residue 176 in puta tive transmembrane helix 4 (M4) because this is an aspartic acid in ra bbit and asparagine in human. Asp-176 in rabbit SGLT1 was replaced wit h asparagine and alanine residues, and the wild-type and mutant protei ns were expressed in Xenopus laevis oocytes. A two-electrode voltage c lamp was used to measure the kinetics of charge transfer. There was no difference between the wild-type and D176N, but there was a 60-mV neg ative shift in the tau/V and Q/V curves with D176A. This suggests that polar residues at position 176 play an important role in determining charge transfer, probably by electrostatic bonding to a neighboring po lar residue in the membrane domain of the protein. The similarity betw een rabbit SGLT1 and the D176N mutant further indicates that other mem brane residues account for the difference between rabbit and human SGL T1. There were only modest changes in the steady-state Na+/glucose cot ransport kinetics between wild-type and D176A mutant transporters in t he voltage range +50 to -50 mV. Model simulations show that the mutati on alters the rate constants for conformational changes of the unloade d transporter. Phlorizin, a specific competitive inhibitor of sugar tr ansport, has a lower affinity for the D176A mutant than for SGLT1. Thi s indicates that polar residues at position 176 hydrogen bond with the -OH group on the B-phenyl ring of the inhibitor.