Neutralization of Conservative Charged Transmembrane Residues in the Na+/Glucose Cotransporter SGLT1

• Published in: Biochemistry (Easton) 1998-07, Vol.37 (29), p.10522-10528
• Main Authors: Panayotova-Heiermann, Mariana, Loo, Donald D. F, Lam, Jason T, Wright, Ernest M
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Alanine - metabolism; Amino Acids - genetics; Amino Acids - metabolism; Animals; Aspartic Acid - genetics; Aspartic Acid - metabolism; Binding, Competitive; Biological Transport - drug effects; Glucose - metabolism; Glutamic Acid - genetics; Glutamic Acid - metabolism; Kinetics; Lysine - genetics; Lysine - metabolism; Membrane Glycoproteins - antagonists & inhibitors; Membrane Glycoproteins - metabolism; Models, Molecular; Monosaccharide Transport Proteins - antagonists & inhibitors; Monosaccharide Transport Proteins - metabolism; Mutagenesis, Site-Directed; Patch-Clamp Techniques; Phlorhizin - pharmacology; Protein Structure, Secondary; Rabbits; Sodium - metabolism; Sodium-Glucose Transporter 1
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9800395

Our goal was to identify pairs of charged residues in the membrane domains of the Na+/glucose cotransporter (SGLT1) that form salt bridges, to obtain information about packing of the transmembrane helices. The strategy was to neutralize Glu225, Asp273, Asp294, and Lys321 in helices 6−8, express the mutants in oocytes, measure [14C]-αMDG uptake, and then attempt to find second-site mutations of opposite charge that restored function. αMDG uptake by E225A was identical to that by SGLT1, whereas transport was reduced by over 90% for D273A, D294A, and K321A and was not restored in the double mutants D273A/K321A or D294A/K321A. This suggested that K321 did not form salt bridges with D273 or D294 and that E225 was not involved in salt-bridging. Neutralization of K321 dramatically changed the Na+ uniport and Na+/glucose cotransport kinetics. The maximum rate of uniport in K321A increased 3−5-fold with a decrease in the apparent affinity for Na+ ( 70 vs 3 mM) and no change in apparent H+ affinity ( 0.5 μM). The change in Na+ affinity caused a +50 mV shift in the charge/voltage (Q/V) and relaxation time constant (τ)/voltage curves in the presteady-state kinetics. The presteady-state kinetics in H+ remained unchanged. The lower Na+ affinity resulted also in a 200-fold increase in the apparent K 0.5 for αMDG and phlorizin. Replacements of K321 with alanine, valine, glutamine, arginine, or glutamic acid residues changed the steady-state kinetics in a similar way. Therefore, we suggest that K321 determines, directly or indirectly, (i) the rate and selectivity of SGLT1 uniport activity and (ii) the apparent affinities of SGLT1 for Na+, and indirectly sugar in the cotransport mode.