Neutralization of conservative charged transmembrane residues in the Na+/glucose cotransporter SGLT1.
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]-alphaMDG uptake, and then attempt to find second-site mutations of opposite charge that restored function. alphaMDG 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 microM). The change in Na+ affinity caused a +50 mV shift in the charge/voltage (Q/V) and relaxation time constant (tau)/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 K0.5 for alphaMDG 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.
Department of Physiology, University of California at Los Angeles Medical Center 90095-1751, USA. mariana@physiology.medsch.ucla.edu
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D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D005947:Glucose / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010695:Phlorhizin / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D051269:Sodium-Glucose Transporter 1
D000596:Amino Acids; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D051269:Sodium-Glucose Transporter 1; D001224:Aspartic Acid; D018698:Glutamic Acid; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose; D008239:Lysine; D000409:Alanine