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...

Rabbit conjunctival epithelium exhibits UTP-dependent Cl(-) secretion into the tears. We investigated whether fluid secretion also takes place. Short-circuit current (I(sc)) was 14.9 +/- 1.4 microA/cm(2) (n = 16). Four P2Y(2) purinergic receptor agonists [UTP and the novel compounds INS365, INS306, and INS440 (Inspire Pharmaceuticals)] added apically (10 microM) resulted in temporary (approximately 30 min) I(sc) increases (88%, 66%, 57%, and 28%, respectively; n = 4 each). Importantly, the conjunctiva transported fluid from serosa to mucosa at a rate of 6.5 +/- 0.7 microl x h(-1) x cm(-2) (range 2.1--15.3, n = 20). Fluid transport was stimulated by mucosal additions of 10 microM: 1) UTP, from 7.4 +/- 2.3 to 10.7 +/- 3.3 microl x h(-1) x cm(-2), n = 5; and 2) INS365, from 6.3 +/- 1.0 to 9.8 +/- 2.5 microl. h(-1) x cm(-2), n = 5. Fluid transport was abolished by 1 mM ouabain (n = 5) and was drastically inhibited by 300 microM quinidine (from 6.4 +/- 1.2 to 3.6 +/- 1.0 microl x h(-1) x cm(-2), n = 4). We conclude that this epithelium secretes fluid actively and that P2Y(2) agonists stimulate both Cl(-) and fluid secretions.

The mechanism of transepithelial fluid transport remains unclear. The prevailing explanation is that transport of electrolytes across cell membranes results in local concentration gradients and transcellular osmosis. However, when transporting fluid, the corneal endothelium spontaneously generates a locally circulating current of approximately 25 microA cm(-2), and we report here that electrical currents (0 to +/-15 microA cm(-2)) imposed across this layer induce fluid movements linear with the currents. As the imposed currents must be approximately 98% paracellular, the direction of induced fluid movements and the rapidity with which they follow current imposition (rise time < or =3 sec) is consistent with electro-osmosis driven by sodium movement across the paracellular pathway. The value of the coupling coefficient between current and fluid movements found here (2.37 +/- 0.11 microm cm(2) hr(-1) microA (-1), suggests that: 1) the local endothelial current accounts for spontaneous transendothelial fluid transport; 2) the fluid transported becomes isotonically equilibrated. Ca(++)-free solutions or endothelial damage eliminate the coupling, pointing to the cells and particularly their intercellular junctions as a main site of electro-osmosis. The polycation polylysine, which is expected to affect surface charges, reverses the direction of current-induced fluid movements. Fluid transport is proportional to the electrical resistance of the ambient medium. Taken together, the results suggest that electro-osmosis through the intercellular junctions is the primary process in a sequence of events that results in fluid transport across this preparation.

Water transport across plasma membranes is a universal property of cells, but the route of such transport is unclear. In this study, volume changes of cells of the J774 murine macrophage-like cell line were monitored by recording the intensity of light scattered by the cells. We investigated the effects of several inhibitors of glucose transport on cell membrane osmotic water permeability as calculated from the rates of cell volume change. Cytochalasin B (2.5 ,ug/ml), phloretin (20 FM), and tomatine (3 jzM) reversibly blocked glucose uptake into these cells. All three inhibitors reversibly decreased the osmotic water permeability of J774 cells from 89.6 ± 3.2 to 27.2 ± 1.4 ,um/sec. We conclude that a major component of the osmotic water flow across the plasma membranes of these cells is accounted for by water traversing their glucose transporters.Water transport across plasma membranes is thought to occur both by diffusion across the lipid bilayer and through specific channels. Although the lipid bilayer of cell membranes has a relatively low intrinsic permeability to water, it may suffice for water exchanges in most cells (1). Some cells [i.e., urinary tract epithelial cells (2) and erythrocytes (3, 4)] display osmotic water permeabilities (P) much higher [up to 350 ,um/sec (5)] than can be accounted for by osmotic water passage through lipid bilayers [P = 4-37 gm/sec (6)], suggesting that they possess membrane channels specialized for water transport. In the human erythrocyte, Solomon et al. (3) proposed that the anion transporter (band 3) serves as a water channel. However, other possible transmembrane water routes in this cell have been suggested (4, 7). Jung et al. (8) used the kinetics of hydrogen-tritium exchange in erythrocytes to suggest that the glucose transporter contains a water pore. Experimental data and theoretical analysis from others (9-11) also suggest that the dimensions and physical properties of the glucose transporter are consistent with its functioning as a water channel. Nonetheless, direct evidence identifying the water channel has been lacking.The identification of water channels in cells has been hindered by the lack of inhibitors that selectively block water transport. We report here that a specific inhibitor of glucose transport [cytochalasin B (CytB)] (12) reversibly slows osmotic water transport across the plasma membranes of J774 cells, a murine macrophage-like cell line (13). For these experiments, we used the intensity of light scattered (Is) by cells to monitor transient cell volume changes. From our evidence, we conclude that glucose transporters serve as the major water channel in these cells. Some of our initial results have appeared in abstract form (14). METHODSCell Culture. J774 cells were maintained as suspension cultures in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% (vol/vol) heat-inactivated calf serum (GIBCO). Cells were plated and maintained overnight as adherent cultures in the above medium on either 12-mm round coverslips (for solut...