Abstract
Site-directed mutagenesis and chemical modification of specific cysteine amino acid side chains by methanethiosulfonate (MTS) derivatives were combined to elucidate structure/function relationships of the cloned rabbit Na+/glucose cotransporter, SGLT1. Each amino acid in the region (residues 162-173) between putative transmembrane helices IV and V of SGLT1 was replaced individually with Cys. Mutant proteins were expressed in Xenopus laevis oocytes and studied using the two-electrode voltage clamp method. At certain key positions, Cys substitution resulted in 1) a change in the apparent affinity for sugar, 2) an alteration in the voltage dependence of the transient currents, and 3) a sensitivity to inhibition by either the ethylamine (MTSEA) or the ethylsulfonate MTS derivatives. For the three Cys mutants inhibited by MTSEA (F163C, A166C, and L173C), inhibition of steady state transport is related to changes in membrane potential-dependent transitions within the Na+/glucose transport cycle. MTSEA shifted the transient currents of these Cys mutants toward more negative membrane potentials (DeltaV0. 5 = -18 mV for F163C and A166C, -12 mV for L173C). When the mutations were combined to produce double and triple Cys mutants, the degree to which the transient currents were shifted along the membrane potential axis by MTSEA correlated with the number of cysteines. In this way it was possible to manipulate the voltage dependence of the transient currents over a range spanning 91 mV. Examination of the Na+ dependence of the transient currents indicates that a 91-mV shift is equivalent to that caused by a 10-fold reduction in the external Na+ concentration. We conclude that this region has a role in determining the Na+ binding- and voltage-sensing properties of SGLT1 and that it forms an alpha-helix with one surface possibly lining a Na+ pore within SGLT1.
Highlights
The Naϩ/glucose cotransporter, SGLT1, utilizes the Naϩ electrochemical gradient to drive transport of sugar across intestinal and renal brush border membranes
For the three Cys mutants inhibited by MTSEA (F163C, A166C, and L173C), inhibition of steady state transport is related to changes in membrane potential-dependent transitions within the Na؉/glucose transport cycle
The schematic is based on a proposed secondary structure in which SGLT1 is composed of a short extracellular N terminus followed by 14 ␣-helical hydrophobic domains that traverse the membrane in zig-zag fashion [33]
Summary
The Naϩ/glucose cotransporter, SGLT1, utilizes the Naϩ electrochemical gradient to drive transport of sugar across intestinal and renal brush border membranes. In addition to SGLT1, many other cotransporters and exchangers that move cations such as Naϩ and Hϩ across lipid bilayers have been found to demonstrate transport activities that are strongly influenced by membrane potential (9 –13) In recent years, this membrane potential dependence of transport has been investigated using “voltage jump” experiments that measure transient charge movements associated with transporter expression [4, 10, 14]. The transient currents exhibited by SGLT1 have been studied using the cut open oocyte method [25], which allows for an ultrafast voltage clamp and access to the intracellular ion concentrations Using this technique, the transient currents were shown to arise from at least three transitions [26]. The distribution of the cysteine mutants that were sensitive to MTS derivatives suggests that this region forms an ␣-helix, one surface of which lines a Naϩ pore within SGLT1
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