Na+/glucose cotransporters (SGLTs) are expressed in the small intestine and the proximal renal tubule, where they play a central role in the absorption of glucose and galactose from food and the reabsorption of glucose from the glomerular filtrate. The regulation of intestinal sugar absorption occurs over two distinct time scales, one over days and the other over minutes. This review focuses on the mechanisms involved in the shorter-term regulation. Recent studies of the mouse intestine in vitro demonstrated that Na+/glucose cotransport is increased two- to eightfold within minutes by the application of forskolin, an agent that increases intracellular cyclic AMP levels. Here we explore how cyclic AMP may upregulate Na+/glucose cotransport. Our strategy was to express cloned SGLT1s in Xenopus laevis oocytes and then use electrophysiological methods to measure (i) the kinetics of Na+/glucose cotransport, (ii) the number of cotransporters in the plasma membrane, and (iii) the net rate of exo- and endocytosis before and after activation of protein kinases. To evaluate the role of cotransporter phosphorylation, we have examined the effect of protein kinase activation on various SGLT1 isoforms and other cotransporters. In oocytes expressing rabbit SGLT1, the activation of protein kinase A (PKA) increased the maximum rate of Na+/glucose cotransport by 30%, and the activation of protein kinase C (PKC) decreased the maximum rate of transport by 60%. Changes in maximum transport rates were accompanied by proportional changes in the number of cotransporters in the plasma membrane and by changes in the area of the membrane. We conclude that PKA and PKC regulate rabbit SGLT1 activity by modulating the number of cotransporters in the plasma membrane and that this occurs through regulation of exocytosis and endocytosis. Given the size of intracellular transport vesicles containing SGLT1, 100-120 nm in diameter, and the density of cotransporters in these vesicles, 10-20 per vesicle, we estimate that the net rate of SGLT1 vesicle exocytosis is about 10,000 s-1 and that this rate increases 100-fold after activation of PKA. The effect of PKA is independent of the presence or absence of consensus sites for phosphorylation on SGLT1. Surprisingly, the effects of PKA or PKC depend critically on the sequence of the contransporter being expressed in the oocyte, e.g. activation of PKC inhibited rabbit and rat SGLT1, but stimulated human SGLT1. This dependency suggests that the regulation of vesicle trafficking by protein kinases depends upon the structure of the cotransporter expressed in the oocyte. Similar considerations apply to other classes of cotransporters, such as the neurotransmitter and dipeptide cotransporters. Our working hypothesis is that the regulation of cotransporter expression by protein kinases occurs largely by regulated exo- and endocytosis, and that the effect of the protein kinases is indirect and determined by critical domains in the cotransporter.
Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices. They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps. The facilitative and H(+)-driven sugar transporters belong to a gene family with a distinctive secondary structure profile. The Na(+)-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels. It is likely that the Na(+)- and H(+)-driven sugar cotransporters share common transport mechanisms. To explore these mechanisms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT) in Xenopus laevis oocytes and measured the kinetics of sugar transport using two-electrode voltage-clamp techniques. For SGLT1, we have developed a six-state ordered model that accounts for the experimental data. To test the model we have carried out the following experiments. (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques. In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage-dependent conformational changes of the protein. Time constants for the carrier currents give estimates of rate constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane. Ultrastructural studies have confirmed these estimates of SGLT1 density. (ii) We have perturbed the kinetics of the cotransporter by site-directed mutagenesis of selected residues.(ABSTRACT TRUNCATED AT 250 WORDS)