A New Human NHERF1 Mutation Decreases Renal Phosphate Transporter NPT2a Expression by a PTH-Independent Mechanism

Background: The sodium-hydrogen exchanger regulatory factor 1 (NHERF1) binds to the main renal phosphate transporter NPT2a and to the parathyroid hormone (PTH) receptor. We have recently identified mutations in NHERF1 that decrease renal phosphate reabsorption by increasing PTH-induced cAMP production in the renal proximal tubule. Methods: We compared relevant parameters of phosphate homeostasis in a patient with a previously undescribed mutation in NHERF1 and in control subjects. We expressed the mutant NHERF1 protein in Xenopus Oocytes and in cultured cells to study its effects on phosphate transport and PTH-induced cAMP production. Results: We identified in a patient with inappropriate renal phosphate reabsorption a previously unidentified mutation (E68A) located in the PDZ1 domain of NHERF1.We report the consequences of this mutation on NHERF1 function. E68A mutation did not modify cAMP production in the patient. PTH-induced cAMP synthesis and PKC activity were not altered by E68A mutation in renal cells in culture. In contrast to wild-type NHERF1, expression of the E68A mutant in Xenopus oocytes and in human cells failed to increase phosphate transport. Pull down experiments showed that E68A mutant did not interact with NPT2a, which robustly interacted with wild type NHERF1 and previously identified mutants. Biotinylation studies revealed that E68A mutant was unable to increase cell surface expression of NPT2a. Conclusions: Our results indicate that the PDZ1 domain is critical for NHERF1- NPT2a interaction in humans and for th

Functional Interaction between CFTR and the Sodium-Phosphate Co-Transport Type 2a in Xenopus laevis Oocytes

A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF) when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a) functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi) was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC) had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis

Analysis of CLCNKB mutations at dimer‐interface, calcium‐binding site, and pore reveals a variety of functional alterations in ClC‐Kb channel leading to Bartter syndrome

International audiencePathological missense mutations in CLCNKB gene give a wide spectrum of clinical phenotypes in Bartter syndrome type III patients. Molecular analysis of the mutated ClC-Kb channels can be helpful to classify the mutations according to their functional alteration. We investigated the functional consequences of nine mutations in the CLCNKB gene causing Bartter syndrome. We first established that all tested mutations lead to decreased ClC-Kb currents. Combining electrophysiological and biochemical methods in Xenopus laevis oocytes and in MDCKII cells, we identified three classes of mutations. One class is characterized by altered channel trafficking. p.A210V, p.P216L, p.G424R, and p.G437R are totally or partially retained in the endoplasmic reticulum. p.S218N is characterized by reduced channel insertion at the plasma membrane and altered pH-sensitivity; thus, it falls in the second class of mutations. Finally, we found a novel class of functionally inactivated mutants normally present at the plasma membrane. Indeed, we found that p.A204T alters the pH-sensitivity, p.A254V abolishes the calcium-sensitivity. p.G219C and p.G465R are probably partially inactive at the plasma membrane. In conclusion, most pathogenic mutants accumulate partly or totally in intracellular compartments, but some mutants are normally present at the membrane surface and simultaneously show a large range of altered channel gating properties

ANP-stimulated Na+ secretion in the collecting duct prevents Na+ retention in the renal adaptation to acid load.

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl- cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis

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

International audienceThe 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+

In silico model of the human ClC-Kb chloride channel: pore mapping, biostructural pathology and drug screening

International audienceThe human ClC-Kb channel plays a key role in exporting chloride ions from the cytosol and is known to be involved in Bartter syndrome type 3 when its permeation capacity is decreased. The ClC-Kb channel has been recently proposed as a potential therapeutic target to treat hypertension. In order to gain new insights into the sequence-structure-function relationships of this channel, to investigate possible impacts of amino-acid substitutions, and to design novel inhibitors, we first built a structural model of the human ClC-Kb channel using comparative modeling strategies. We combined in silico and in vitro techniques to analyze amino acids involved in the chloride ion pathway as well as to rationalize the possible role of several clinically observed mutations leading to the Bartter syndrome type 3. Virtual screening and drug repositioning computations were then carried out. We identified six novel molecules, including 2 approved drugs, diflusinal and loperamide, with Kd values in the low micromolar range, that block the human ClC-Kb channel and that could be used as starting point to design novel chemical probes for this potential therapeutic target

NPT2a and CFTR expressed in <i>X. laevis</i> oocytes co-immunoprecipitate.

<p>Representative experiment showing the co-immunoprecipitation of NPT2a and CFTR after their expression in <i>X. laevis</i> oocytes. Proteins from oocytes co-expressing Myc-NPT2a and CFTR were immunoprecipitated with MM13-4 Ab, or with IgG1 Ab used as a negative control. They were probed with the anti-Myc antibody. The molecular weight of the detected protein is indicated Similar results were obtained in N  =  3 independent experiments.</p

Effects of agonists of EPAC and PKA pathways on NPT2a function and surface expression.

<p>Phosphate-induced currents (IPi) were evoked in voltage-clamped (–50 mV) cells by the exposure of NPT2a-oocytes (white columns), or NPT2a+CFTR-oocytes (black columns) to 1 mM Pi. As above, IPi was first induced in basal condition, ^basalIPi, then after the activation of a cAMP-dependent signaling pathway, ^exptlIPi, during 10 min (panels A and B). Results are as means ± SEM. IPi were normalized against ^basalIPi from NPT2a-oocytes from the same batch of oocytes. The significance of the difference between ^basalIPi from NPT2a-oocytes and ^basalIPi or ^exptlIPi from NPT2a+CFTR-oocytes was analyzed using unpaired Student’s <i>t</i>-test (*: P < 0.05). The significance of the difference between ^basalIPi and ^exptlIPi within the same type of oocytes was assessed using paired Student’s <i>t</i>-test (#: P < 0.05). <u>A</u>: Effect of para-Chlorophenylthio-2′-O-methyladenosine-3′, 5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP, 25 µM), an activator on the EPAC pathway on IPi in NPT2a- and NPT2a+CFTR-oocytes (n  =  9 for each type; N =  2). <u>B</u>: Effect of N⁶-monobutyryladenosine-3′, 5′-cyclic monophosphate (6-MB-cAMP, 25 µM), an activator of the PKA pathway on IPi in NPT2a- and NPT2a+CFTR-oocytes (n  =  19 and n  =  22, respectively; N =  6).</p

Phosphate-induced current in <i>X. laevis</i> oocytes.

<p>Phosphate-induced current (1 mM of Pi added in ND96) was measured at holding potential (Vc) of −50 mV in oocytes expressing NPT2a or CFTR alone or co-expressing NPT2a and CFTR; oocytes injected with water were used as control oocytes. <u>A</u>: Original tracings showing the current induced by the addition of 1 mM Pi (indicated by black bars) in the superfusing medium. The type of oocytes is indicated above the tracings. <u>B</u>: Summary of the results (means ± SEM) of the effect of 1 mM Pi in the different types of oocytes (indicated below the columns). Significance of the results *: P<0.05</p