Jean-Yves Lapointe

Macula densa cell signaling involves ATP release through a maxi anion channel

Macula densa cells are unique renal biosensor cells that detect changes in luminal NaCl concentration ([NaCl]L) and transmit signals to the mesangial cell/afferent arteriolar complex. They are the critical link between renal salt and water excretion and glomerular hemodynamics, thus playing a key role in regulation of body fluid volume. Since identification of these cells in the early 1900s, the nature of the signaling process from macula densa cells to the glomerular contractile elements has remained unknown. In patch–clamp studies of macula densa cells, we identified an [NaCl]L-sensitive ATP-permeable large-conductance (380 pS) anion channel. Also, we directly demonstrated the release of ATP (up to 10 μM) at the basolateral membrane of macula densa cells, in a manner dependent on [NaCl]L, by using an ATP bioassay technique. Furthermore, we found that glomerular mesangial cells respond with elevations in cytosolic Ca2+ concentration to extracellular application of ATP (EC50 0.8 μM). Importantly, we also found increases in cytosolic Ca2+ concentration with elevations in [NaCl]L, when fura-2-loaded mesangial cells were placed close to the basolateral membrane of macula densa cells. Thus, cell-to-cell communication between macula densa cells and mesangial cells, which express P2Y2 receptors, involves the release of ATP from macula densa cells via maxi anion channels at the basolateral membrane. This mechanism may represent a new paradigm in cell-to-cell signal transduction mediated by ATP.

Identification of a novel Na+/myo-inositol cotransporter.

rkST1, an orphan cDNA of the SLC5 family (43% identical in sequence to the sodium myo-inositol cotransporter SMIT), was expressed in Xenopus laevis oocytes that were subsequently voltage-clamped and exposed to likely substrates. Whereas superfusion with glucose and other sugars produced a small inward current, the largest current was observed with myo-inositol. The expressed protein, which we have named SMIT2, cotransports myo-inositol with aK m of 120 μm and displays a current-voltage relationship similar to that seen with SMIT (now called SMIT1). The transport is Na+-dependent, with aK m of 13 mm. SMIT2 exhibits phlorizin-inhibitable presteady-state currents and substrate-independent “Na+ leak” currents similar to those of related cotransporters. The steady-state cotransport current is also phlorizin-inhibitable with a K i of 76 μm. SMIT2 exhibits stereospecific cotransport of bothd-glucose and d-xylose but does not transport fucose. In addition, SMIT2 (but not SMIT1) transportsd-chiro-inositol. Based on previous publications, the tissue distribution of SMIT2 is different from that of SMIT1, and the existence of this second cotransporter may explain much of the heterogeneity that has been reported for inositol transport.

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.

Electrogenic amino acid exchange via the rBAT transporter

A cDNA clone was isolated from rabbit renal cortex using DNA‐mediated expression cloning, which caused alanine‐dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na‐independent amino acid transporter previously cloned elsewhere. Exposure of cDNA‐injected oocytes to neutral amino acids led to voltage‐dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut‐open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.

Fast voltage clamp discloses a new component of presteady-state currents from the Na(+)-glucose cotransporter

The human Na(+)-glucose cotransporter (hSGLT1) has been shown to generate, in the absence of sugar, presteady-state currents in response to a change in potential, which could be fitted with single exponentials once the voltage had reached a new constant value. By the cut-open oocyte technique (voltage rising-speed approximately 1 mV/microsecond), phlorizin-sensitive transient currents could be detected with a higher time resolution during continuous intracellular perfusion. In the absence of sugar and internal Na+, and with 90 mM external Na+ concentration ([Na+]o), phlorizin-sensitive currents exhibited two relaxation time-constants: tau 1 increased from 2 to 10 ms when Vm decreased from +60 mV to -80 mV and remained at 10 ms for more negative Vm; tau 2 ranged from 0.4 to 0.8 ms in a weakly voltage-dependent manner. According to a previously proposed model, these two time constants could be accounted for by 1) Na+ crossing a fraction of the membrane electrical field to reach its binding site on the carrier and 2) conformational change of the free carrier. To test this hypothesis, the time constants were measured as [Na+]o was progressively reduced to 0 mM. At 30 and 10 mM external Na+, tau 1 reached the same plateau value of 10 ms but at more negative potentials (-120 and -160 mV, respectively). Contrary to the prediction of the model, two time constants continued to be detected in the bilateral absence of Na+ (at pH 8.0). Under these conditions, tau 1 continuously increased through the whole voltage range and did not reach the 10 ms level even when Vm had attained -200 mV while tau 2 remained in the range of 0.4-0.8 ms. These results indicate that 1) conformational change of the free carrier across the membrane must occur in more than one step and 2) Na+ binding/debinding is not responsible for either of the two observed exponential components of transient currents. By use of the simplest kinetic model accounting for the portion of the hSGLT1 transport cycle involving extracellular Na+ binding/debinding and the dual-step conformational change of the free carrier, tau 1 and tau 2 were fitted throughout the voltage range, and a few sets of parameters were found to reproduce the data satisfactorily. This study shows that 1) tau 1 and tau 2 correspond to two steps in the conformational change of the free carrier, 2) Na+ binding/debinding modulates the slow time constant (tau 1) and 3) a voltage-independent slow conformational change of the free carrier accounts for the observed plateau value of 10 ms.

Pharmacological and Biochemical Evidence for the Regulation of Osteocalcin Secretion by Potassium Channels in Human Osteoblast‐like MG–63 Cells

Previous reports have suggested the involvement of voltage‐activated calcium (Ca2+) channels in bone metabolism and in particular on the secretion of osteocalcin by osteoblast‐like cells. 1 We now report that potassium (K+) channels can also modulate the secretion of osteocalcin by MG–63 cells, a human osteosarcoma cell line. When 1,25‐dihydroxyvitamin D3(1,25(OH)2D3)‐treated MG–63 cells were depolarized by step increases of the extracellular K+ concentration ([K+]out) from 5–30 mM, osteocalcin (OC) secretion increased from a control value of 218 ± 13 to 369 ± 18 ng/mg of protein/48 h (p < 0.005 by analysis of variance). In contrast, in the absence of 1,25(OH)2D3, there is no osteocalcin secretion nor any effect of cell depolarization on this activity. The depolarization‐induced increase in 1,25(OH)2D3‐dependent osteocalcin secretion was totally inhibited in the presence of 10 μM Nitrendipine (a Ca2+ channel blocker, p < 0.005) without affecting cellular alkaline phosphatase nor cell growth. Charybdotoxin, a selective blocker of Ca2+‐dependent K+ channels (maxi‐K) present in MG–63 cells, 2 stimulated 1,25(OH)2D3‐induced osteocalcin synthesis about 2‐fold (p < 0.005) after either 30, 60, or 120 minutes of treatment. However, Charybdotoxin was without effect on basal release of osteocalcin in the absence of 1,25(OH)2D3 pretreatment. Using patch clamp technique, we occasionally observed the presence of a small conductance K+ channel, compatible with an ATP‐dependent K+ channel (GKATP) in nonstimulated cells, whereas multiple channel openings were observed when cells were treated with Diazoxide, a sulfonamide derivative which opens GKATP. Western blot analysis revealed the presence of the N‐terminal peptide of GKATP in MG–63 cells, and its expression was regulated with the proliferation rate of these cells, maximal detection by Western blots being observed during the logarithmic phase of the cycle. Glipizide and Glybenclamide, selective sulfonylureas which can block GKATP, dose‐dependently enhanced 1,25(OH)2D3‐induced OC secretion (p < 0.005). Reducing the extracellular calcium concentration with EGTA (μM range) totally inhibited the effect of Glipizide and Glybenclamide on osteocalcin secretion (p < 0.005), which remained at the same levels as controls. Diazoxide totally prevented the effect of these sulfonylureas. These results suggest that voltage‐activated Ca2+ channels triggered via cell depolarization can enhance 1,25(OH)2D3‐induced OC release by MG–63 cells. In addition, OC secretion is increased by blocking two types of K+ channels: maxi‐K channels, which normally hyperpolarize cells and close Ca2+ channels, and GKATP channels. The role of these channels is closely linked to the extracellular Ca2+ concentration.

Determination of transport stoichiometry for two cation-coupled myo-inositol cotransporters : SMIT2 and HMIT

Three different mammalian myo‐inositol cotransporters are currently known; two are Na+‐coupled (SMIT1 and SMIT2) and one is proton‐coupled (HMIT). Although their transport stoichiometries have not been directly determined, significant cooperativities in the Na+ activation of SMIT1 and SMIT2 suggest that more than one Na+ ion drives the transport of each myo‐inositol. The two techniques used here to determine transport stoichiometry take advantage of the electrogenicity of both SMIT2 and HMIT expressed in Xenopus oocytes. The first method compares the measurement of charge transferred into voltage‐clamped oocytes with the simultaneous uptake of radiolabelled substrate. The second approach uses high accuracy volume measurements to determine the transport‐dependent osmolyte uptake and compares it to the amount of charge transported. This method was calibrated using a potassium channel (ROMK2) and was validated with the Na+/glucose cotransporter SGLT1, which has a known stoichiometry of 2 : 1. Volume measurements indicated a stoichiometric ratio of 1.78 ± 0.27 ion per α‐methyl‐glucose (αMG) for SGLT1 whereas the radiotracer uptake method indicated 2.14 ± 0.05. The two methods yielded a SMIT2 stoichiometry measurement of 1.75 ± 0.30 and 1.82 ± 0.10, both in agreement with a 2 Na+:1 myo‐inositol stoichiometry. For HMIT, the flux ratio was 1.02 ± 0.04 charge per myo‐inositol, but the volumetric method suggested 0.67 ± 0.05 charge per myo‐inositol molecule. This last value is presumed to be an underestimate of the true stoichiometry of one proton for one myo‐inositol molecule due to some proton exchange for osmotically active species. This hypothesis was confirmed by using SGLT1 as a proton‐driven glucose cotransporter. In conclusion, despite the inherent difficulty in estimating the osmotic effect of a proton influx, the volumetric method was found valuable as it has the unique capacity of detecting unidentified transported substrates.

Activation of Maxi-K Channels by Parathyroid Hormone and Prostaglandin E2 in Human Osteoblast Bone Cells

Abstract.Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mm Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nm parathyroid hormone (PTH), 5 nm prostaglandin E2 (PGE2) or 0.1 mm dibutyryl cAMP + 1 μm forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mm K) the channel has a conductance of 246 ± 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (PK/PNa) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (Po) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the Po vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mm ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE2 or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (45Ca++) by MG-63 cells was stimulated in the presence of PTH and PGE2, an effect inhibited by Nitrendipine (10 μm). Second, whereas PGE2 stimulated the calcium-activated maxi-K channel in 2 mm Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE2 did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mm Ca Ringer and 10 μm Nitrendipine in CAP configuration, PGE2 (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE2 or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx.