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

Abstract

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.

Mechanistic Insights and Functional Determinants of the Transport Cycle of the Ascorbic Acid Transporter SVCT2

“…The kinetic analysis does not provide enough data to identify the specific step in the overall transport cycle that is affected by the presence of Na ϩ . Data obtained with the Na ϩ -glucose and the Na ϩ -glutamate co-transporters was interpreted as indicating that the Na ϩ effect on the transport K m is associated with conformational transitions that expose the substrate binding site (22,23). However, recent data with the Lac permease, a transporter of lactose whose transport activity is driven by the 3-min uptake assay in the presence of 135 mM Na ϩ , choline ϩ , Li ϩ , Cs ϩ , or K ϩ in medium containing 100 M ascorbic acid.…”

Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding

“…The cysteine mutations were generated via the megaprimer protocol of polymerase chain reaction mutagenesis as described previously and confirmed by sequencing (21).…”

“…NH 2-terminal myc-tagged WT and mutants used for COS-7 transfections were prepared as described previously (21). Nontransfected cells and COS-7 cells transfected with vector alone served as controls.…”

“…We then studied the apparent competition between phloridzin and MTSEA with respect to MTSEA reactivity with cysteine-156 (21). COS-7 cells expressing WT SGLT1 and T156C were each reacted with biotin-MTSEA.…”

Imaging of the Dynamics of Glucose Uptake in the Cytosol of COS-7 Cells by Fluorescent Nanosensors

“…COS-7 cells express the glucose transporter GLUT1 (40). Pharmacological studies indicate that active transport via sodium/glucose cotransporter does not contribute significantly to glucose uptake (41). Hexokinase rather than glucokinase is used for glucose phosphorylation (32).…”