Although vitamin C is critical to human physiology, it is not clear how it is taken up into cells. The kinetics of cell and tissue accumulation of ascorbic acid in vitro indicate that the process is mediated by specific transporters at the cell membrane. Some experimental observations have linked the transport of ascorbic acid with hexose transport systems in mammalian cells, although no clear information is available regarding the specific role(s) of these transporters, if any, in this process. Here we use the Xenopus laevis oocyte expression system to show that the mammalian facilitative hexose transporters are efficient transporters of the oxidized form of vitamin C (dehydroascorbic acid). Two transport pathways, one with low affinity and one with high affinity for dehydroascorbic acid, were found in oocytes expressing the mammalian transporters, and these oocytes accumulated vitamin C against a concentration gradient when supplied with dehydroascorbic acid. We obtained similar results in experiments using normal human neutrophils. These observations indicate that mammalian facilitative hexose transporters are a physiologically significant pathway for the uptake and accumulation of vitamin C by cells, and suggest a mechanism for the accumulation of ascorbic acid against a concentration gradient.

Artemisinins are the most important class of antimalarial drugs. They specifically inhibit PfATP6, a SERCA-type ATPase of Plasmodium falciparum. Here we show that a single amino acid in transmembrane segment 3 of SERCAs can determine susceptibility to artemisinin. An L263E replacement of a malarial by a mammalian residue abolishes inhibition by artemisinins. Introducing residues found in other Plasmodium spp. also modulates artemisinin sensitivity, suggesting that artemisinins interact with the thapsigargin-binding cleft of susceptible SERCAs.

The glucose transporters (GLUT/SLC2A) are members of the major facilitator superfamily. Here, we generated a three-dimensional model for Glut1 using a two-step strategy: 1), GlpT structure as an initial homology template and 2), evolutionary homology using glucose-6-phosphate translocase as a template. The resulting structure (PDB No. 1SUK) exhibits a water-filled passageway communicating the extracellular and intracellular domains, with a funnel-like exofacial vestibule (infundibulum), followed by a 15 A-long x 8 A-wide channel, and a horn-shaped endofacial vestibule. Most residues which, by mutagenesis, are crucial for transport delimit the channel, and putative sugar recognition motifs (QLS, QLG) border both ends of the channel. On the outside of the structure there are two positively charged cavities (one exofacial, one endofacial) delimited by ATP-binding Walker motifs, and an exofacial large side cavity of yet unknown function. Docking sites were found for the glucose substrate and its inhibitors: glucose, forskolin, and phloretin at the exofacial infundibulum; forskolin, and phloretin at an endofacial site next to the channel opening; and cytochalasin B at a positively charged endofacial pocket 3 A away from the channel. Thus, 1SUK accounts for practically all biochemical and mutagenesis evidence, and provides clues for the transport process.

Glut-1 deficiency syndrome was first described in 1991 as a sporadic clinical condition, later shown to be the result of haploinsufficiency. We now report a family with Glut-1 deficiency syndrome affecting 5 members over 3 generations. The syndrome behaves as an autosomal dominant condition. Affected family members manifested mild to severe seizures, developmental delay, ataxia, hypoglycorrhachia, and decreased erythrocyte 3-O-methyl-D-glucose uptake. Seizure frequency and severity were aggravated by fasting, and responded to a carbohydrate load. Glut-1 immunoreactivity in erythrocyte membranes was normal. A heterozygous R126H missense mutation was identified in the 3 patients available for testing, 2 brothers (Generation 3) and their mother (Generation 2). The sister and her father were clinically and genotypically normal. In vitro mutagenesis studies in Xenopus laevis oocytes demonstrated significant decreases in the transport of 3-O-methyl-D-glucose and dehydroascorbic acid. Xenopus oocyte membranes expressed high amounts of the R126H mutant Glut-1. Kinetic analysis indicated that replacement of arginine-126 by histidine in the mutant Glut-1 resulted in a lower Vmax. These studies demonstrate the pathogenicity of the R126H missense mutation and transmission of Glut-1 deficiency syndrome as an autosomal dominant trait.

Water traverses the plasma membranes of some eukaryotic cells faster than can be explained by the water permeability of their lipid bilayers. This has led to a search for a water channel. Our previous work identified glucose transporters as candidates for such a channel. We report here that Xenopus laevis oocytes injected with mRNA encoding the brain/Hep G2, adult skeletal muscle/adipocyte, or liver forms of the glucose transporter exhibit an osmotic water permeability of their plasma membranes larger than that of untreated oocytes. The osmotic water permeability component attributable to glucose transporters increased an average of 4.8-fold in the inected oocytes. These studies provide direct evidence that the facilitative, sodium-independent mammalian glucose transporters serve as membrane water channels.Virtually all mammalian cells express proteins that mediate the stereospecific transport of D-glucose across their plasma membranes by facilitated diffusion (1). This mode of transport is characteristic of glucose transporters (GTs), which have been molecularly identified in brain (2, 3), skeletal muscle and adipocytes (4-7), hepatocytes (8, 9), and fetal muscle (10). In addition, several investigators have suggested, on the basis of experimental (11, 12) and theoretical (13) considerations, that the GT may contain a water-filled channel that spans the membrane.We have provided evidence that in the macrophage-like J774 cell line, GTs serve as water channels (14). Our evidence (14) was based on the observation that inhibitors of glucose transport significantly reduce the rate of osmotic water flow across the cell's plasma membrane. In those studies, the osmotic water permeability (P) was monitored by measuring the rate of cell volume change in response to an osmotic challenge. In cells exposed to either hypotonic or hypertonic challenge in the presence of a specific inhibitor of glucose transport, cytochalasin B, P was reduced from 85 ,um/sec to 25 um/sec. The latter value is consistent with the P of lipid bilayers (15).To rigorously test the hypothesis that GTs serve as water channels, we have expressed mammalian GTs in frog oocytes as described by Vera and Rosen (16) and have compared the P value of these oocytes with that of control oocytes.Specifically, oocytes injected with mRNA encoding either brain/Hep G2 GT (GT2), adult skeletal muscle/adipocyte GT (GT1), or liver GT (GT3) exhibit a 20-to 40-fold increase in the rates of uptake of 2-deoxy-D-glucose or 3-0-methylglucose above those values observed in control or uninjected oocytes (16,17). In addition, the uptake of2-deoxy-D-glucose in mRNA-injected oocytes was inhibited by cytochalasin B (16), by phloretin (Phl; data not shown), and by elevated concentrations of D-glucose, but not by L-glucose (16). Thus, mammalian GTs expressed in Xenopus oocytes retain characteristic properties of GTs from mammalian cells.Given this background, we injected Xenopus laevis oocytes with mRNA encoding three different GTs originally cloned from rat brain, adult ske...