Gabrielle Planelles

Functional Characterization of a Calcium-Sensing Receptor Mutation in Severe Autosomal Dominant Hypocalcemia with a Bartter-Like Syndrome

The extracellular Ca(2+)-sensing receptor (CaSR) plays an essential role in extracellular Ca(2+) homeostasis by regulating the rate of parathyroid hormone (PTH) secretion and the rate of calcium reabsorption by the kidney. Activation of the renal CaSR is thought to inhibit paracellular divalent cation reabsorption in the cortical ascending limb (cTAL) both directly and indirectly via a decrease in NaCl transport. However, in patients with autosomal dominant hypocalcemia (ADH), caused by CaSR gain-of-function mutations, a defect in tubular NaCl reabsorption with renal loss of NaCl has not been described so far. This article describes a patient with ADH due to a gain-of-function mutation in the CaSR, L125P, associated with a Bartter-like syndrome that is characterized by a decrease in distal tubular fractional chloride reabsorption rate and negative NaCl balance with secondary hyperaldosteronism and hypokalemia. The kinetics of activation of the L125P mutant receptor expressed in HEK-293 cells, assessed by measuring CaSR-stimulated changes in intracellular Ca(2+) and ERK activity, showed a dramatic reduction in the EC(50) for extracellular Ca(2+) compared with the wild-type and a loss-of-function mutant CaSR (I40F). This study describes the first case of ADH associated with a Bartter-like syndrome. It is herein proposed that the L125P mutation of the CaSR, which represents the most potent gain-of-function mutation reported so far, may reduce NaCl reabsorption in the cTAL sufficiently to result in renal loss of NaCl with secondary hyperaldosteronism and hypokalemia.

NHERF1 mutations and responsiveness of renal parathyroid hormone.

Impaired renal phosphate reabsorption, as measured by dividing the tubular maximal reabsorption of phosphate by the glomerular filtration rate (TmP/GFR), increases the risks of nephrolithiasis and bone demineralization. Data from animal models suggest that sodium-hydrogen exchanger regulatory factor 1 (NHERF1) controls renal phosphate transport. We sequenced the NHERF1 gene in 158 patients, 94 of whom had either nephrolithiasis or bone demineralization. We identified three distinct mutations in seven patients with a low TmP/GFR value. No patients with normal TmP/GFR values had mutations. The mutants expressed in cultured renal cells increased the generation of cyclic AMP (cAMP) by parathyroid hormone (PTH) and inhibited phosphate transport. These NHERF1 mutations suggest a previously unrecognized cause of renal phosphate loss in humans.

NH3Is Involved in the Transport Induced by the Functional Expression of the Human Rh C Glycoprotein

Renal ammonium (NH3 + batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{NH}_{4}^{+}) end{document}) transport is a key process for body acid-base balance. It is well known that several ionic transport systems allow batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{NH}_{4}^{+}) end{document} transmembrane translocation without high specificity batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{NH}_{4}^{+}) end{document}, but it is still debated whether NH3, and more generally, gas, may be transported by transmembrane proteins. The human Rh glycoproteins have been proposed to mediate ammonium transport. Transport of batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{NH}_{4}^{+}) end{document} and/or NH3 by the epithelial Rh C glycoprotein (RhCG) may be of physiological importance in renal ammonium excretion because RhCG is mainly expressed in the distal nephron. However, RhCG function is not yet established. In the present study, we search for ammonium transport by RhCG. RhCG function was investigated by electrophysiological approaches in RhCG-expressing Xenopus laevis oocytes. In the submillimolar concentration range, NH4Cl exposure induced inward currents (IAM) in voltage-clamped RhCG-expressing cells, but not in control cells. At physiological extracellular pH (pHo) = 7.5, the amplitude of IAM increased with NH4Cl concentration and membrane hyperpolarization. The amplitude of IAM was independent of external Na+ or K+ concentrations but was enhanced by alkaline pHo and decreased by acid pHo. The apparent affinity of RhCG for batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{NH}_{4}^{+}) end{document} was affected by NH3 concentration and by changing pHo, whereas the apparent affinity for NH3 was unchanged by pHo, consistent with direct NH3 involvement in RhCG function. The enhancement of methylammonium-induced current by NH3 further supported this conclusion. Exposure to 500 μm NH4Cl induced a biphasic intracellular pH change in RhCG-expressing oocytes, consistent with both NH3 and batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{NH}_{4}^{+}) end{document} enhanced influx. Our results support the hypothesis of a specific role for RhCG in NH3 and batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{NH}_{4}^{+}) end{document} transport.

Rescue of ΔF508-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) by Curcumin: Involvement of the Keratin 18 Network

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, ΔF508, causes retention of ΔF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl– channels in the plasma membrane. ΔF508-CFTR retains some Cl– channel activity so increased expression of ΔF508-CFTR in the plasma membrane can restore Cl– secretion deficiency. Recently, curcumin was shown to rescue ΔF508-CFTR localization and function. In our previous work, the keratin 18 (K18) network was implicated in ΔF508-CFTR trafficking. Here, we hypothesized that curcumin could restore a functional ΔF508-CFTR to the plasma membrane acting via the K18 network. First, we analyzed the effects of curcumin on the localization of ΔF508-CFTR in different cell lines (HeLa cells stably transfected with wild-type CFTR or ΔF508-CFTR, CALU-3 cells, or cystic fibrosis pancreatic epithelial cells CFPAC-1) and found that it was significantly delocalized toward the plasma membrane in ΔF508-CFTR-expressing cells. We also performed a functional assay for the CFTR chloride channel in CFPAC-1 cells treated or not with curcumin and detected an increase in a cAMP-dependent chloride efflux in treated ΔF508-CFTR-expressing cells. The K18 network then was analyzed by immunocytochemistry and immunoblot exclusively in curcumin-treated or untreated CFPAC-1 cells because of their endogenic ΔF508-CFTR expression. After curcumin treatment, we observed a remodeling of the K18 network and a significant increase in K18 Ser52 phosphorylation, a site directly implicated in the reorganization of intermediate filaments. With these results, we propose that K18 as a new therapeutic target and curcumin, and/or its analogs, might be considered as potential therapeutic agents for cystic fibrosis.

Expression of the human erythroid Rh glycoprotein (RhAG) enhances both NH3 and NH4+ transport in HeLa cells

The erythroid Rh-associated glycoprotein (RhAG) is strictly required for the expression of the Rh blood group antigens carried by Rh (D,CE) proteins. A biological function for RhAG in ammonium transport has been suggested by its ability to improve survival of an ammonium-uptake-deficient yeast. We investigated the function of RhAG by studying the entry of NH3/NH4+ in HeLa cells transiently expressing the green fluorescent protein (GFP)-RhAG fusion protein and using a fluorescent proton probe to measure intracellular pH (pHi). Under experimental conditions that reduce the intrinsic Na/H exchanger activity, exposure of control cells to a 10 mM NH4Cl-containing solution induces the classic pHi response profile of cells having a high permeability to NH3 (PNH3) but relatively low permeability to NH4+ (PNH4). In contrast, under the same conditions, the pHi profile of cells expressing RhAG clearly indicated an increased PNH4, as evidenced by secondary reacidification during NH4Cl exposure and a pHi undershoot below the initial resting value upon its removal. Measurements of pHi during methylammonium exposure showed that RhAG expression enhances the influx of both the unprotonated and ionic forms of methylammonium. Using a mathematical model to adjust passive permeabilities for a fit to the pHi profiles, we found that RhAG expression resulted in a threefold increase of PNH4 and a twofold increase of PNH3. Our results are the first evidence that the human erythroid RhAG increases the transport of both NH3 and NH4+.

Further investigation of ionic diffusive properties and of NH4+ pathways in Xenopus laevis oocyte cell membrane.

To study the ionic diffusive properties and the NH4+ pathways in theXenopus laevis oocyte cell membrane, we recorded the effects of various inhibitors on membrane potential (Vm) and membrane resistance (Rm); intracellular acidification was taken as an index of NH4+ influx from the bath to the cytoplasm. The following results were obtained: in the control state, barium and quinine (Q) ions depolarizedVm and raisedRm, consistent with inhibition of K+ conductance(s). Diphenylamine-2-carboxylic acid (DPC), 3′, 5′-dichlorodiphenylamine-2-carboxylic acid (DCDPC) and gadolinium ions hyperpolarizedVm and increasedRm, suggesting the inhibition of nonselective cationic conductance(s). In the presence of 20 mmol/l NH4Cl,Vm depolarized,Rm fell, and intracellular pH (pHi) decreased, consistent with an NH4+ influx. In the presence of DPC, the same manoeuvre induced a biphasicVm change (i.e. a spike depolarization followed by a membrane hyperpolarization) and a fall ofRm; in most oocytes, intracellular acidification persisted and was reversible upon adding ouabain (Oua). These results indicate that a DPC-sensitive conductance is not the unique NH4+ pathway and that Na, K-ATPase may also mediate NH4+ influx. However, Oua did not prevent theRm decrease, suggesting that ouabain-insensitive rheogenic pathway(s) are activated. Thus, we investigated theVm change induced by NH4Cl addition in the presence of DPC: the spike depolarization followed by secondary hyperpolarization became a plateau depolarization when Q was added, suggesting involvement of Q-sensitive pathway(s) in the above described biphasicVm change. In the presence of DPC, Q, and Oua, intracellular acidification upon adding NH4C1 persisted consistent with further NH4+ influx through quinine-, DPC- and Oua-insensitive pathway (s).

Extracellular ATP raises cytosolic calcium and activates basolateral chloride conductance in Necturus proximal tubule

1 Extracellular nucleotides modulate ionic transport mechanisms in various epithelia. In the present study, we investigated the effects of extracellular ATP on the intracellular free Ca2+ concentration ([Ca2+]i) and electrophysiological properties of Necturus maculosus proximal convoluted tubule (PCT). 2 ATP raised [Ca2+]i in microdissected fura‐2‐loaded PCTs (half‐maximal effect, ≈15 μmol l−1 ATP). The initial ATP‐induced changes in [Ca2+]i were not blunted by the removal of external Ca2+ nor by the presence of Ca2+ channel blockers, but were abolished by thapsigargin and suramin. The sequence for the potency of various agonists on [Ca2+]i was 2‐methylthioATP (2MeSATP) = ADP = ATP ≫ UTP, 2′,3′‐O‐(4‐benzoilbenzoil) ATP (BzATP), α,β‐methylene ATP (AMPCPP), adenosine. 3 In vivo electrophysiological measurements showed that 100 μmol l−1 peritubular ATP added to a Ringer solution reduced the basolateral cell membrane potential (Vm) and increased the cell membrane input conductance. In a low Cl− solution, this ATP‐induced depolarization was enhanced. These effects were inhibited by 1 mmol l−1SITS, consistent with the activation of a basolateral Cl− conductance. 4 The ATP‐induced change in Vm was reproduced by ADP but not by UTP or adenosine, and was prevented by suramin. 5 The ATP‐induced membrane depolarization was not influenced by thapsigargin, BAPTA AM, or staurosporin and was not reproduced by manoeuvres increasing [Ca2+]i or intracellular cAMP content. 6 We conclude that, in Necturus PCT, a P2y receptor mobilizes Ca2+ mainly from intracellular pools and increases a basolateral Cl− conductance, GCl. The activation of GCl occurs by a mechanism which is not related either to an increase in [Ca2+]i or cAMP content, or to PKC activation.

Ammonium Homeostasis and Human Rhesus Glycoproteins

The brain ammonium production is detoxified by astrocytes, the gut ammonium production is detoxified by hepatic cells, and the renal ammonium production plays a major role in renal acid excretion. As a result of ammonium handling in these organs, the ammonium and pH values are strictly regulated in plasma. Up until recently, it was accepted that mammalian cell transmembrane ammonium transport was due to NH4+ transport by non-specific transporting systems, and to non-ionic NH3 diffusion, whereas lower organisms (such as bacteria, yeasts and plants) were endowed with specific ammonium transporters (Amts). Sequence homologies between Amts and human Rhesus (Rh) glycoproteins (RhAG, from erythroid cells, and RhBG and RhCG from epithelial cells) raised the hypothesis that Rh glycoproteins act as specific ammonium transporters, further sustained by the polarized distribution of RhBG and RhCG in gut, kidney and liver. Results from functional studies agree that Rh glycoproteins are the first ammonium transporters reported in mammals. However, the nature of the transported specie(s) is much debated: in particular, it is proposed that Rh glycoproteins mediate a direct NH3 transport, or that they mediate an indirect NH3 transport (resulting from NH4+ for H+ exchange). Direct NH3 transport (associated or not with NH4+ transport) raises the exciting hypothesis that Rh glycoproteins may also transport other gasses than NH3 (namely, CO2).

Control of Basal CFTR Gene Expression by Bicarbonate-Sensitive Adenylyl Cyclase in Human Pulmonary Cells

The CFTR protein, encoded by the gene whose mutations induce Cystic Fibrosis, is an anion channel devoted mainly to chloride and bicarbonate transmembrane transport, but which also regulates transport of several other ions. Moreover, it is implicated in the cell response to inflammation, and, reciprocally, cftr gene expression is modulated by inflammatory stimuli and transduction pathways. Looking for a control of CFTR expression by ionic conditions, we investigated the effect of altered extracellular bicarbonate ion concentration on CFTR expression in human pulmonary Calu-3 cells. We found that basal cftr gene transcription is enhanced when extracellular HCO3- concentration increases from 0 to 25 mmol/l. The transduction pathway controlled by these extracellular [HCO3-] variations includes cAMP production linked to the stimulation of soluble adenylyl cyclase (sAC), and nuclear accumulation of the transcription factor, CREB. Basal membrane content in CFTR protein exhibits the same variations as cftr mRNA in cells incubated in the presence of extracellular [HCO3-] between 0 and 25 mmol/l, and is also decreased by inhibiting sAC in the presence of HCO3-. These results show that bicarbonate-controlled sAC stimulation must be taken into account in cell physiology and that basal CFTR expression depends on an ionic parameter.

Chloride transport in the renal proximal tubule

The renal proximal tubule is responsible for most of the renal sodium, chloride, and bicarbonate reabsorption. Micropuncture studies and electrophysiological techniques have furnished the bulk of our knowledge about the physiology of this tubular segment. As a consequence of the leakiness of this epithelium, paracellular ionic transport—in particular that of Cl−—is of considerable importance in this first part of the nephron. It was long accepted that proximal Cl− reabsorption proceeds solely paracellularly, but it is now known that transcellular Cl− transport also exists. Cl− channels and Cl−-coupled transporters are involved in transcellular Cl− transport. In the apical membrane, Cl−/anion (formate, oxalate and bicarbonate) exchangers represent the first step in transcellular Cl− reabsorption. A basolateral Cl−/HCO3− exchanger, involved in HCO3− reclamation, participates in the rise of intracellular Cl− activity above its equilibrium value, and thus also contributes to the creation of an outwardly directed electrochemical Cl− gradient across the cell membranes. This driving force favours Cl− diffusion from the cell to the lumen and to the interstitium. In the basolateral membrane, the main mechanism for transcellular Cl− reabsorption is a Cl− conductance, but a Na+-driven Cl−/HCO3− exchanger may also participate in Cl− reabsorption.