Research projects: : Function and dysfunction of transport systems
Cell function and homeostasis are dependent on cell membrane transport systems. The latter mediate the transmembrane translocation of various substrates; thanks to fine regulation, the transport rate is adjusted to fulfil the specialized function of the cell, of the organ and to maintain body homeostasis. In order to decipher the normal function of transport systems (when still unknown), to study their regulation, and to evaluate the functional consequences of their mutations (detected in patients), we bolster their heterologous expression in Xenopus laevis oocyte, a convenient model for functional studies.
Expression is obtained after micro-injection of RNAs encoding for a given transporter. Oocytes possess all the material necessary for the synthesis, maturation, and trafficking of heterologous proteins. The expression level is evaluated by biochemical and molecular approaches; function is assessed using electrophysiological approaches (including ion selective microelectrodes) and labelled fluxes.
Expression of GFP-fused protein (RhCG) in X. laevis oocyte.
Measurement of intracellular pH in an oocyte supplemented with carbonic anhydrase type2 (CAII): effect of a challenging CO2 gas level (in kPa). CO2 exposure induces cell acidification, due to CO2 diffusion into the cell and the subsequent dissociation of its hydrated form into bicarbonate and H+, thus the observed acidification. The oocyte was supplemented with CAII to accelerate the intracellular reaction.
Measurement of creatine-induced current in voltage-clamped oocytes (-50 mV) expressing SLC26 A8 (Na-creatine cotransport). WT: wild-type; P1, P2: mutated SLC26A8 isolated from 2 patients with mental retardation. Whereas creatine, 500 µM, induced a large inward current (due to Na+ influx into the cell) in oocytes expressing the WT Na-creatine cotransport, this manoeuvre had no effect in oocytes expressing the P1 or the P2 mutated transporters, consistent with total loss of function. SLC26A8 is expressed in kidney and brain. Both P1 and P2 patients were diagnosed due to severe mental retardation.
There is a tight link between ionic transport systems and the cellular metabolic status. Our current project in renal physiology is to investigate the effect of the AMPK/mTor pathway on various renal transport systems (including Na-creatine cotransport, the H, K-ATPase type 2, and the ammonium transporter RhCG) during stress conditions (i.e., exposure to renal toxic solutes -such as Cd2+ or albumin - and change in O2 gas levels). The studies include pharmacological activation/inhibition, heterologous expression of the kinase (WT, constitutively active or dead kinase). Subsequent changes in transport function are assessed using electrophysiological approaches.
Gabrielle Planelles: DR2 Inserm
Naziha Bakouh: Post doctoral fellow
Fatna-Léa Makaci : Technical assistant
Valayannopoulos* V, Bakouh* N, Mazzuca M, Nonnenmache L, Hubert L, Makaci FL, Chabli A , Salomons GA, Mellot-Draznieks C, Brulé E, De Lonlay P, Toulhoat H, Munnich A, Planelles G, DE Keyzer Y. Functional and electrophysiological characterization of four non-truncating mutations responsible for creatine transporter (SLC6A8) deficiency syndrome. J Inh Metab Dis 36:103-112. 2013 *equal contribution. PMID: 22644605
Bakouh N, Bienvenu T, Thomas A, Ehrenfeld J, Liote H, Roussel D, Duquesnoy P, Farman N, Viel
M, Cherif-Zahar B, Amselem S, Taam RA, Edelman A, Planelles G, Sermet-Gaudelus I. Characterization of SLC26A9 in Patients with CF-Like Lung Disease. Hum Mutat.34(10):1404-14. 2013.
Bakouh N, Cherif-Zahar B, Hulin P, Prie D, Friedlander G, Edelman A, Planelles G. Functional interaction between CFTR and
the sodium-phosphate co-transport type 2a in Xenopus laevis oocytes. PLoSONE 7(4):e34879. 2012. PMID: 22514683
Rotmann A, Sanchez C, Guiguemde A, Rohobach P, Dave A, Bakouh N, Planelles G, Lanzer M. PfCHA is a mitochondrial divalent cation/H+ antiporter in Plasmodium falciparum. Mol Microbiol.76(6):1591-606. 2010. PMID: 20487273
Bakouh N, Benjelloun F, Hulin
P, Edelman A, Cherif-Zahar B, Planelles G. NH3 is involved in NH4+ transport induced by the functional expression of the human RhCG glycoprotein. J Biol Chem. 279:15975-83, 2004.