Functional studies of the rabbit intestinal Na+/glucose carrier (SGLT1) expressed in COS-7 cells: evaluation of the mutant A166C indicates this region is important for Na+-activation of the carrier.
We have exploited two mutants of the rabbit intestinal Na+/glucose carrier SGLT1 to explore the structure/function relationship of this Na+/glucose transporter in COS-7 cells. A functional N-terminal myc-epitope-tagged SGLT1 protein was constructed and used to determine the plasma-membrane localization of SGLT1. The kinetic and specificity characteristics of the myc-tagged SGLT1 mutant were identical with those of wild-type SGLT1. Immunogold labelling and electron microscopy confirmed the topology of the N-terminal region to be extracellular. Expression of the SGLT1 A166C mutant in these cells showed diminished levels of Na+-dependent alpha-methyl-d-glucopyranoside transport activity compared with wild-type SGLT1. For SGLT1 A166C, Vmax was 0.92+/-0.08 nmol/min per mg of protein and Km was 0.98+/-0.13 mM; for wild-type SGLT1, Vmax was 1.98+/-0.47 nmol/min per mg of protein and Km was 0.36+/-0.16 mM. Significantly, phlorrhizin (phloridzin) binding experiments confirmed equal expression of Na+-dependent high-affinity phlorrhizin binding to COS-7 cells expressing SGLT1 A166C or wild-type SGLT1 (Bmax 1.55+/-0.18 and 1.69+/-0.57 pmol/mg of protein respectively); Kd values were 0.46+/-0.15 and 0.51+/-0.11 microM for SGLT1 A166C and wild-type SGLT1 respectively. The specificity of sugar interaction was unchanged by the A166C mutation. We conclude that the replacement of an alanine residue by cysteine at position 166 has a profound effect on transporter function, resulting in a decrease in transporter turnover rate by a factor of 2. Taken as a whole the functional changes observed by SGLT1 A166C are most consistent with the mutation having caused an altered Na+ interaction with the transporter.
Vayro
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Medical Research Council (M.R.C.) Membrane Biology Group, Department of Medicine, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8 Canada.
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Silverman
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D000818:Animals; D001692:Biological Transport / Q000502:physiology; D019556:COS Cells; D003545:Cysteine / Q000235:genetics; D007422:Intestines / Q000502:physiology*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D008757:Methylglucosides / Q000493:pharmacokinetics; D016253:Microscopy, Immunoelectron; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000502:physiology*; D009154:Mutation / Q000235:genetics; D010695:Phlorhizin / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000494:pharmacology*; D051269:Sodium-Glucose Transporter 1; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014162:Transfection / Q000235:genetics
D008562:Membrane Glycoproteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D051269:Sodium-Glucose Transporter 1; C027020:methylglucoside; D012964:Sodium; D010695:Phlorhizin; D003545:Cysteine