Our group focuses on the molecular mechanisms controlling the membrane trafficking of renal ion transporters. Regulation of ion transport activity engages a tightly controlled delivery of intracellular pools of transport proteins to the plasma membrane and /or the endocytic retrieval of transport proteins from the cell surface. Similar to other classes of membrane proteins, transport proteins possess sequence motives that function as sorting signals. These signals are interpreted and acted upon through protein-protein interactions with components of the cellular sorting machinery. Cells can also exploit such interactions in order to control transport activity by regulating the trafficking of transport proteins between ER-Golgi system, intracellular vesicular storage pools and the plasma membrane. The identifications and the characterizations of protein-protein interactions in a cell type are necessary for a better understanding of its functions.
In the recent years, we mainly focused on the identification of the molecular switches that regulate kidney specific Na-K-2Cl co-transporter, NKCC2. Loss-of function mutations in the NKCC2 gene cause type I Bartter’s syndrome, a life threatening disease featuring arterial hypotension along with electrolyte abnormalities. Yet the molecular mechanisms by which this apical cotransporter is regulated in health and disease have remained poorly understood,mainly because of the difficulty in expressing the co-transporter protein in mammalian cells. Our group was the first to express NKCC2 protein in mammalian cell, thereby providing a powerful tool to study the molecular mechanisms underlying the intracellular trafficking of NKCC2, particularly those based on the co-transporter’s interactions with other proteins. To completely understand the functional regulation of NKCC2, we must unveil the molecular determinants underlying membrane trafficking of NKCC2 in a mammalian cell background, and understand how these regulatory factors orchestrate the journey of the co-transporter from the moment of its synthesis until its export to the cell surface.
We seek to understand:
1/ how NKCC2 proteins sort and target to appropriate sites within cells.
2/ how abnormalities in NKCC2 protein trafficking lead to Bartter’s syndrome.
Using the yeast two hybrid system and co-immunorecipitation assays, we have identified several NKCC2 interacting partners. For each NKCC2-partner interaction, we propose to elucidate, in vitro and in vivo, the functional consequences of the interaction on NKCC2 surface expression and activity. Accordingly, efforts will be made to move beyond studies using heterologous expression systems to more physiologically relevant conditions (such as animal models) to further identify the protein interaction networks and processes governing protein transport and function of relevance to renal health and disease. A better understanding of the regulatory pathways acting on NKCC2 activity and expression would help to gain insights into the pathophysiology of salt and water retention, in fine, would offer new possible target for pharmaceutical approach to prevent and/or treat kidney disorders related to sodium and/or water balance.
Kamel Laghmani, CR1 CNRS
Sylvie Demaretz, Engineer
Eli Seaayfan, M2 student
Polyhydramnios, Transient Antenatal Bartter’s Syndrome, and MAGED2 Mutations.
Kamel Laghmani, Bodo B. Beck, Sung-Sen Yang, Elie Seaayfan, Andrea Wenzel, Björn Reusch, Helga Vitzthum, Dario Priem, Sylvie Demaretz, Klasien Bergmann, Leonie K. Duin, Heike Göbel, Christoph Mache, Holger Thiele, Malte P. Bartram, Carlos Dombret, Janine Altmüller, Peter Nürnberg, Thomas Benzing, Elena Levtchenko, Hannsjörg W. Seyberth, Günter Klaus, Gökhan Yigit, Shih-Hua Lin, Albert Timmer, Tom J. de Koning, Sicco A. Scherjon, Karl P. Schlingmann, Mathieu J.M. Bertrand, Markus M. Rinschen, Olivier de Backer, Martin Konrad, Martin Kömhoff. N Engl J Med. 2016. May 12;374(19):1853-1863. PMID: 27120771
OS9 interacts with NKCC2 and targets its immature form for the endoplasmic-reticulum-associated. Seaayfan E, Defontaine N, Demaretz S, Zaarour N, Laghmani K . J Biol Chem. 2015 Dec 31. PMID: 26721884.
Effects of NKCC2 isoform regulation on NaCl transport in thick ascending limb and macula densa: a modeling study. Edwards A, Castrop H, Laghmani K, Vallon V, Layton AT. Effects of NKCC2 isoform regulation on NaCl transport in thick ascending limb and macula densa: a modeling study. Am J Physiol Renal Physiol. 2014 Jul 15;307(2):F137-46. PMCID: PMC4101627.
Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression. Trudu M, Janas S, Lanzani C, Debaix H, Schaeffer C, Ikehata M, Citterio L, Demaretz S, Trevisani F, Ristagno G, Glaudemans B, Laghmani K, Dell'Antonio G; Swiss Kidney Project on Genes in Hypertension (SKIPOGH) team, Loffing J, Rastaldi MP, Manunta P, Devuyst O, Rampoldi L. Nat Med. 2013 Dec;19(12):1655-60.: PMC3856354.
SPAK differentially mediates vasopressin effects on sodium cotransporters. Saritas T, Borschewski A, McCormick JA, Paliege A, Dathe C, Uchida S, Terker A, Himmerkus N, Bleich M, Demaretz S, Laghmani K, Delpire E, Ellison DH, Bachmann S, Mutig K. . J Am Soc Nephrol. 2013 Feb;24(3):407-18. PMCID: PMC3582200.
Multiple Evolutionarily Conserved Di-Leucine Like Motifs in the Carboxyl Terminus Control the Anterograde Trafficking of NKCC2. Zaarour N, Demaretz S, Defontaine N, Zhu Y, Laghmani K. J Biol Chem. 2012. Dec 14; 287(51):42642-53. PMID: 23105100
PTH-independent regulation of blood calcium concentration by the calcium-sensing receptor. Loupy A, Ramakrishnan SK, Wootla B, Chambrey R, de la Faille R, Bourgeois S, Bruneval P, Mandet C, Christensen EI, Faure H, Cheval L, Laghmani K, Collet C, Eladari D, Dodd RH, Ruat M, Houillier P. J Clin Invest. 2012 Sep 4; 122(9): 3355-67. PMID: 22886306
Regulation of pendrin by cAMP: possible involvement in b-adrenergic-dependent NaCl retention. Azroyan A, Morla L, Crambert G, Laghmani K, Ramakrishnan S, Edwards A, Doucet A. . Am J Physiol Renal Physiol. 2012 May 1; 302(9): F1180-7. PMID: 22262479
Heterogeneity in the processing of CLCN5 mutants related to Dent disease. Grand T, L'Hoste S, Mordasini D, Defontaine N, Keck M, Pennaforte T, Genete M, Laghmani K, Teulon J, Lourdel S. Hum Mutat. 2011 Apr; 32(4): 476-83. PMID: 21305656
Secretory carrier membrane protein 2 regulates exocytic insertion of NKCC2 into the cell membrane. Zaarour N, Defontaine N, Demaretz S, Azroyan A, Cheval L, Laghmani K. J Biol Chem. 2011. Mar 18; 286(11): 9489-502. PMID: 21205824
Regulation of pendrin by pH: dependence on glycosylation. Azroyan A, Laghmani K, Crambert G, Mordasini D, Doucet A, Edwards A. Biochem J. 2011 . Feb 15; 434(1): 61-72. PMID: 21073444
A Highly Conserved Motif at the C-terminus Dictates ER exit and Cell-Surface Expression of NKCC2. Zaarour N, Demaretz S, Defontaine, Mordasini D, Laghmani K. J Biol Chem. 2009 Aug 7; 284(32):21752-64. PMID: 19535327
NKCC2 surface expression in mammalian cells: Down-regulation by novel interaction with aldolase B. Benziane B, Demaretz S, Defontaine N, Zaarour N, Cheval L, Bourgeois S, Klein C, Froissart M, Blanchard A, Paillard M, Gamba G, Houillier P, Laghmani K. J Biol Chem. 2007 . Nov 16; 282(46): 33817-30. PMID: 17848580