FUNCTIONAL-STUDIES OF THE RABBIT INTESTINAL NA+ GLUCOSE CARRIER (SGLT1) EXPRESSED IN COS-7 CELLS - EVALUATION OF THE MUTANT A166C INDICATESTHIS REGION IS IMPORTANT FOR NA+-ACTIVATION OF THE CARRIER/

We have exploited two mutants of the rabbit intestinal Na+/glucose car rier SGLT1 to explore the structure/function relationship of this Na+/ glucose transporter in COS-7 cells. A functional N-terminal myc-epitop e-tagged SGLT1 protein was constructed and used to determine the plasm a-membrane localization of SGLT1. The kinetic and specificity characte ristics of the myc-tagged SGLT1 mutant were identical with those of wi ld-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 N a+-dependent alpha-methyl-D-glucopyranoside transport activity compare d with wild-type SGLT1. For SGLT1 A166C, V-max, was 0.92+/-0.08 nmol/m in per mg of protein and K-m was 0.98+/-0.13 mM; for wild-type SGLT1, V-max was 1.98+/-0.47 nmol/min per mg of protein and K-m was 0.36+/-0. 16 mM. Significantly, phlorrhizin (phloridzin) binding experiments con firmed equal expression of Na+-dependent high-affinity phlorrhizin bin ding to COS-7 cells expressing SGLT1 A166C or wild-type SGLT1 (B-max 1 .55+/-0.18 and 1.69+/-0.57 pmol/mg of protein respectively); K-d value s were 0.46+/-0.15 and 0.51+/-0.11 mu M 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 transpor ter function, resulting in a decrease in transporter turnover rate by a factor of 2. Taken as a whole the functional changes observed by SGL T1 A166C are most consistent with the mutation having caused an altere d Na+ interaction with the transporter.