It recently was proposed [Loo, D. D. F., Zeuthen, T., Chandy, G. & Wright, E. M. (1996) Proc. Natl. Acad. Sci. USA 93, 13367-13370] that SGLT1, the high affinity intestinal and renal sodium͞glucose cotransporter carries water molecules along with the cosubstrates with a strict stoichiometry of two Na ؉ , one glucose, and Ϸ220 water molecules per transport cycle. Using electrophysiology together with sensitive volumetric measurements, we investigated the nature of the driving force behind the cotransporter-mediated water flux. The osmotic water permeability of oocytes expressing human SGLT1 (L p ؎ SE) averaged 3.8 ؎ 0.3 ؋ 10 ؊4 cm⅐s ؊1 (n ‫؍‬ 15) and addition of 100 M phlorizin (a specific SGLT1 inhibitor) reduced the permeability to 2.2 ؎ 0.2 ؋ 10 ؊4 cm⅐s ؊1 (n ‫؍‬ 15), confirming the presence of a significant water permeability closely associated with the cotransporter. Addition of 5 mM ␣-methylglucose (␣MG) induced an average inward current of 800 ؎ 10 nA at ؊50 mV and a water influx reaching 120 ؎ 20 pL cm ؊2 ⅐s ؊1 within 5-8 min. After rapidly inhibiting the Na ؉ ͞glucose cotransport with phlorizin, the water flux remained significantly elevated, clearly indicating the presence of a local osmotic gradient (⌬) estimated at 16 ؎ 2 mOsm. In short-term experiments, a rapid depolarization from ؊100 to 0 mV in the presence of ␣MG decreased the cotransport current by 94% but failed to produce a comparable reduction in the swelling rate. A mathematical model depicting the intracellular accumulation of transported osmolytes can accurately account for these observations. It is concluded that, in SGLT1-expressing oocytes, ␣MG-dependent water influx is induced by a local osmotic gradient by using both endogenous and SGLT1-dependent water permeability.

The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa membrane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17-SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mutation (c.176+1G>A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na/H exchanger 3, and to signaling pathways, such as the A-kinase anchor protein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters.

The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) Ca V channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca V 1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly 432 and Gly 436 in Ca V 1.2) to form a triglycine motif unique to HVA Ca V channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca 2؉ channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca V 1.2, ␣-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with Ca V 1.2 wild type > G770A > G422A Х G436A Ͼ Ͼ G432A (from the fastest to the slowest). Mutations at position Gly 432 produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly 436 to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX 3 G residues blunted Ca 2؉ -dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca V 2.3, mutation of the distal glycine Gly 352 impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX 3 G motif in the voltage-dependent activation and inactivation gating of HVA Ca V channels with the distal glycine residue being mostly involved in the inactivation gating.Voltage-dependent calcium channels are membrane-bound proteins that form large aqueous pores for the selective diffusion of Ca 2ϩ ions across the plasma membrane (1, 2). Native Ca 2ϩ channels are composed of the pore-forming Ca V ␣1, the disulfur-linked dimer Ca V ␣2␦, the intracellular Ca V ␤ subunits (␤1-␤4), and in some cases the Ca V ␥ subunit (3). To date, molecular cloning has identified the primary structures for 10 distinct calcium channel Ca V ␣ 1 subunits (1, 4 -9) that are classified into three main subfamilies according to their gating properties (Ca V 1, Ca V 2, and Ca V 3). Whereas all voltage-gated Ca 2ϩ channel ␣1 subunits activate and inactivate in response to membrane depolarization, the high voltage-activated (HVA) 2 Ca V 1 and Ca V 2 ␣1 subunits operate at markedly more positive membrane potentials than low voltage-activated Ca V 3 channel ␣1 subunits.In the absence of a crystal structure for these proteins, details regarding the structural determinants of the channel inner pore as well as the molecular mechanism underlying the activation of Ca V ␣1 subunits remain sketchy. Structural studies have revealed that the architecture of the ion-selective pore is conserved in the homologous ␣ subunit of different K ϩ channels (10 -15) with the ...

The recent demonstration that the human colon adenocarcinoma cell line Caco-2 was susceptible to spontaneous enterocytic differentiation led us to consider the question as to whether Caco-2 cells would exhibit sodium-coupled transport of sugars. This problem was investigated using isotopic tracer flux measurements of the nonmetabolizable sugar analog alpha-methylglucoside (AMG). AMG accumulation in confluent monolayers was inhibited to the same extent by sodium replacement, 200 microM phlorizin, 1 mM phloretin, and 25 mM D-glucose, but was not inhibited further in the presence of both phlorizin and phloretin. Kinetic studies were compatible with the presence of both a simple diffusive process and a single, Na+-dependent, phlorizin- and phloretin-sensitive AMG transport system. These results also ruled out any interaction between AMG and a Na+-independent, phloretin-sensitive, facilitated diffusion pathway. The brush-border membrane localization of the Na+-dependent system was inferred from the observations that its functional differentiation was synchronous with the development of brush-border membrane enzyme activities and that phlorizin and phloretin addition 1 hr after initiating sugar transport produced immediate inhibition of AMG uptake as compared to ouabain. Finally, it was shown that brush-border membrane vesicles isolated from the human fetal colonic mucosa do possess a Na+-dependent transport pathway(s) for D-glucose which was inhibited by AMG and both phlorizin and phloretin. Caco-2 cells thus appear as a valuable cell culture model to study the mechanisms involved in the differentiation and regulation of intestinal transport functions.

Over the last decade, several cotransport studies have led to the proposal of secondary active transport of water, challenging the dogma that all water transport is passive. The major observation leading to this interpretation was that a Na+ influx failed to reproduce the large and rapid cell swelling induced by Na+/solute cotransport. We have investigated this phenomenon by comparing a Na+/glucose (hSGLT1) induced water flux to water fluxes triggered either by a cationic inward current (using ROMK2 K+ channels) or by a glucose influx (using GLUT2, a passive glucose transporter). These proteins were overexpressed in Xenopus oocytes and assayed through volumetric measurements combined with double-electrode electrophysiology or radioactive uptake measurements. The osmotic gradients driving the observed water fluxes were estimated by comparison with the swelling induced by osmotic shocks of known amplitude. We found that, for equivalent cation or glucose uptakes, the combination of substrate accumulations observed with ROMK2 and GLUT2 are sufficient to provide the osmotic gradient necessary to account for a passive water flux through SGLT1. Despite the fact that the Na+/glucose stoichiometry of SGLT1 is 2:1, glucose accumulation accounts for two-thirds of the osmotic gradient responsible for the water flux observed at t = 30 s. It is concluded that the different accumulation processes for neutral versus charged solutes can quantitatively account for the fast water flux associated with Na+/glucose cotransport activation without having to propose the presence of secondary active water transport.