Pierre Bissonnette

Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon

SummaryThe 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 α-methylglucoside (AMG). AMG accumulation in confluent monolayers was inhibited to the same extent by sodium replacement, 200 μm phlorizin, 1mm phloretin, and 25mm 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) ford-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.

Local osmotic gradients drive the water flux associated with Na+/glucose cotransport

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 sodium/glucose cotransporter carries water molecules along with the cosubstrates with a strict stoichiometry of two Na+, one glucose, and ≈220 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 (Lp ± SE) averaged 3.8 ± 0.3 × 10−4 cm⋅s−1 (n = 15) and addition of 100 μM phlorizin (a specific SGLT1 inhibitor) reduced the permeability to 2.2 ± 0.2 × 10−4 cm⋅s−1 (n = 15), confirming the presence of a significant water permeability closely associated with the cotransporter. Addition of 5 mM α-methyl-glucose (αMG) induced an average inward current of 800 ± 10 nA at −50 mV and a water influx reaching 120 ± 20 pL cm−2 ⋅s−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 (Δπ) estimated at 16 ± 2 mOsm. In short-term experiments, a rapid depolarization from −100 to 0 mV in the presence of αMG 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, αMG-dependent water influx is induced by a local osmotic gradient by using both endogenous and SGLT1-dependent water permeability.

Glucose Accumulation Can Account for the Initial Water Flux Triggered by Na+/Glucose Cotransport

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.

Functional expression of tagged human Na+—glucose cotransporter in Xenopus laevis oocytes

1 High‐affinity, secondary active transport of glucose in the intestine and kidney is mediated by an integral membrane protein named SGLT1 (sodium glucose cotransporter). Though basic properties of the transporter are now defined, many questions regarding the structure‐ function relationship of the protein, its biosynthesis and targeting remain unanswered. In order to better address these questions, we produced a functional hSGLT1 protein (from human) containing a reporter tag. 2 Six constructs, made from three tags (myc, haemaglutinin and poly‐His) inserted at both the C‐ and N‐terminal positions, were thus tested using the Xenopus oocyte expression system via electrophysiology and immunohistochemistry. Of these, only the hSGLT1 construct with the myc tag inserted at the N‐terminal position proved to be of interest, all other constructs showing no or little transport activity. A systematic comparison of transport properties was therefore performed between the myc‐tagged and the untagged hSGLT1 proteins. 3 On the basis of both steady‐state (affinities for substrate (glucose) and inhibitor (phlorizin) as well as expression levels) and presteady‐state parameters (transient currents) we conclude that the two proteins are functionally indistinguishable, at least under these criteria. Immunological detection confirmed the appropriate targeting of the tagged protein to the plasma membrane of the oocyte with the epitope located at the extracellular side. 4 The myc‐tagged hSGLT1 was also successfully expressed in polarized MDCK cells. α‐Methylglucose uptake studies on transfected cells showed an exclusively apical uptake pathway, thus indicating that the expressed protein was correctly targeted to the apical domain of the cell. 5 These comparative studies demonstrate that the myc epitope inserted at the N‐terminus of hSGLT1 produces a fully functional protein while other epitopes of similar size inserted at either end of the protein inactivated the final protein.

The Role of the GX9GX3G Motif in the Gating of High Voltage-activated Ca2+ Channels

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) CaV channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in CaV1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly432 and Gly436 in CaV1.2) to form a triglycine motif unique to HVA CaV channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca2+ channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In CaV1.2, α-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 CaV1.2 wild type > G770A > G422A ≅ G436A >> G432A (from the fastest to the slowest). Mutations at position Gly432 produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly436 to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX3G residues blunted Ca2+-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In CaV2.3, mutation of the distal glycine Gly352 impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX3G motif in the voltage-dependent activation and inactivation gating of HVA CaV channels with the distal glycine residue being mostly involved in the inactivation gating.

The effects of melatonin on Ca2+ homeostasis in endothelial cells

The effect of melatonin on the Ca2+ signaling process in bovine aortic endothelial cells (BAE) and in primary cultured vascular endothelial cells from normotensive Sprague Dawley (SDR) and genetically hypertensive (SHR) rats was investigated using the Ca2+ indicator Fura‐2. Acute applications of melatonin failed to initiate a Ca2+ response in the three cell types considered. However, preincubating SHR aortic endothelial cells with exposure to melatonin increased the internal Ca2+ release triggered by bradykinin (BK) and ATP while stimulating the related agonist‐evoked Ca2+ entry. This effect appeared specific for SHR cells, as a similar incubation period failed to alter the Ca2+ responses in BAE and SDR cells. Because of the known overproduction of free radicals in SHR cells, the effect of melatonin on Ca2+ signaling was also tested in SDR and BAE cells exposed to the superoxide anion radical. Melatonin reversed the deleterious action of free radicals on Ca2+ signaling in both cases, suggesting that its stimulatory effect in SHR was linked to its antioxidative properties. Finally, experiments where melatonin was applied between successive BK stimulation periods showed an enhancement of the agonist‐evoked Ca2+ entry in BAE and SDR cells. This effect appeared to be independent of the production of second messengers as no specific binding sites for melatonin, including MT1, MT2 and MT3 receptors, could be detected in BAE cells. We conclude that melatonin improves Ca2+ signaling in dysfunctional endothelial cells characterized by an overproduction of free radicals while stimulating the agonist‐evoked Ca2+ entry in normal endothelial cells through a mechanism not related to its antioxidative properties.

Identification of a Disulfide Bridge Linking the Fourth and the Seventh Extracellular Loops of the Na+/Glucose Cotransporter

The Na+/glucose cotransporter (SGLT1) is an archetype for the SLC5 family, which is comprised of Na+-coupled transporters for sugars, myo-inositol, choline, and organic anions. Application of the reducing agent dithriothreitol (DTT, 10 mM) to oocytes expressing human SGLT1 affects the proteins presteady-state currents. Integration of these currents at different membrane potentials (Vm) produces a Q-V curve, whose form was shifted by +25 mV due to DTT. The role of the 15 endogenous cysteine residues was investigated by expressing SGLT1 constructs, each bearing a single mutation for an individual cysteine, in Xenopus oocytes, using two-microelectrode voltage-clamp electrophysiology and fluorescent labeling. 12 of the 15 mutants were functional and could be separated into three distinct groups based on the effect of the mutation on the Q-V curve: four mutants did not perturb the transferred charge, six mutants shifted the Q-V curve towards negative potentials, and two mutants (C255A and C511A) produced a shift in the positive direction that was identical to the shift produced by DTT on the wild-type (wt) SGLT1. The double mutant C255,511A confirms that the effects of each single mutant on the Q-V curve were not additive. With respect to wt SGLT1, the apparent affinities for α-methylglucose (αMG) were increased in a similar manner for the single mutants C255A and C511A, the double mutant C255,511A as well as for wt SGLT1 treated with DTT. When exposed to a maleimide-based fluorescent probe, wt SGLT1 was not significantly labeled but mutants C255A and C511A could be clearly labeled, indicating an accessible cysteine residue. These residues are presumed to be C511 and C255, respectively, as the double mutant C255,511A could not be labeled. These results strongly support the hypothesis that C255 and C511 form a disulfide bridge in human SGLT1 and that this disulfide bridge is involved in the conformational change of the free carrier.

Effects of hyperosmolarity on the Na+-myo-inositol cotransporter SMIT2 stably transfected in the Madin-Darby canine kidney cell line

Myo-inositol (MI) is a compatible osmolyte used by cells to compensate for changes in the osmolarity of their surrounding milieu. In kidney, the basolateral Na(+)-MI cotransporter (SMIT1) and apical SMIT2 proteins are homologous cotransporters responsible for cellular uptake of MI. It has been shown in the Madin-Darby canine kidney (MDCK) cell line that SMIT1 expression was under the control of the tonicity-sensitive transcription factor, tonicity-responsive enhancer binding protein (TonEBP). We used an MDCK cell line stably transfected with SMIT2 to determine whether variations in external osmolarity could also affect SMIT2 function. Hyperosmotic conditions (+200 mosM raffinose or NaCl but not urea) generated an increase in SMIT2-specific MI uptake by three- to ninefold in a process that required protein synthesis. Using quantitative RT-PCR, we have determined that hyperosmotic conditions augment both the endogenous SMIT1 and the transfected SMIT2 mRNAs. Transport activities for both SMIT1 and SMIT2 exhibited differences in their respective induction profiles for both their sensitivities to raffinose, as well as in their time course of induction. Application of MG-132, which inhibits nuclear translocation of TonEBP, showed that the effect of osmolarity on transfected SMIT2 was unrelated to TonEBP, unlike the effect observed with SMIT1. Inhibition studies involving the hyperosmolarity-related MAPK suggested that p38 and JNK play a role in the induction of SMIT2. Further studies have shown that hyperosmolarity also upregulates another transfected transporter (Na(+)-glucose), as well as several endogenously expressed transport systems. This study shows that hyperosmolarity can stimulate transport in a TonEBP-independent manner by increasing the amount of mRNA derived from an exogenous DNA segment.

Expression of the sodium–myo‐inositol cotransporter SMIT2 at the apical membrane of Madin‐Darby canine kidney cells

Myo‐inositol is a compatible osmolyte used by cells which are challenged by variations in extracellular osmolarity, as in the renal medulla. In order to accumulate large quantities of this polyol, cells rely on Na+‐dependent transporters such as SMIT1. We have recently identified a second Na+–myo‐inositol cotransporter, SMIT2, which presents transport characteristics corresponding to those recently described for the apical membrane of renal proximal tubules. In order to further characterize this transport system, we transfected Madin‐Darby canine kidney (MDCK) cells with rabbit SMIT2 cDNA and selected a stable clone with a high expression level. The accumulation of radiolabelled myo‐inositol by this cell line is 20‐fold larger than that seen in native MDCK cells. The affinity for myo‐inositol of MDCK cells transfected with SMIT2 is slightly lower (Km= 334 μm) than that found in voltage‐clamped Xenopus laevis oocytes expressing SMIT2 (Km= 120 μm). Transport studies performed using semipermeable filters showed complete apical targeting of the SMIT2 transporter. This apical localization of SMIT2 was confirmed by transport studies on purified rabbit renal brush border membrane vesicles (BBMVs). Using a purified antibody against SMIT2, we were also able to detect the SMIT2 protein (molecular mass = 66 kDa) in Western blots of BBMVs purified from SMIT2‐transfected MDCK cells. SMIT2 activity was also shown to be stimulated 5‐fold when submitted to 24 h hypertonic treatment (+200 mosmol l−1). The SMIT2‐MDCK cell line thus appears to be a promising model for studying SMIT2 biochemistry and regulation.

Characterization of D150E and G196D aquaporin-2 mutations responsible for nephrogenic diabetes insipidus: importance of a mild phenotype.

Aquaporin-2 (AQP2) is a water channel responsible for the final water reabsorption in renal collecting ducts. Alterations in AQP2 function induce nephrogenic diabetes insipidus (NDI), a condition characterized by severe polyuria and polydipsia. Three patients affected with severe NDI, who were compound heterozygous for the AQP2 mutations D150E and G196D, are presented here along with a mildly affected D150E homozygous patient from another family. Using Xenopus oocytes as an expression system, these two mutations (G196D and D150E) were compared with the wild-type protein (AQP2-wt) for functional activity (water flux analysis), protein maturation, and plasma membrane targeting. AQP2-wt induces a major increase in water permeability (P(f) = 47.4 +/- 12.2 x 10(-4) cm/s) whereas D150E displays intermediate P(f) values (P(f) = 12.5 +/- 3.0 x 10(-4) cm/s) and G196D presents no specific water flux, similar to controls (P(f) = 2.1 +/- 0.8 x 10(-4) cm/s and 2.2 +/- 0.7 x 10(-4) cm/s, respectively). Western blot and immunocytochemical evaluations show protein targeting that parallels activity levels with AQP2-wt adequately targeted to the plasma membrane, partial targeting for D150E, and complete sequestration of G196D within intracellular compartments. When coinjecting AQP2-wt with mutants, no (AQP2-wt + D150E) or partial (AQP2-wt + G196D) reduction of water flux were observed compared with AQP2-wt alone, whereas complete loss of function was found when both mutants were coinjected. These results essentially recapitulate the clinical profiles of the family members, showing a typical dominant negative effect when G196D is coinjected with either AQP2-wt or D150E but not between AQP2-wt and D150E mutant.