Luc Caron

Cloning and Functional Characterization of a Cation-Cl− Cotransporter-interacting Protein

To date, the cation-Cl−cotransporter (CCC) family comprises two branches of homologous membrane proteins. One branch includes the Na+-K+-Cl− cotransporters (NKCCs) and the Na+-Cl− cotransporter, and the other branch includes the K+-Cl− cotransporters. Here, we have isolated the first member of a third CCC family branch. This member shares ∼25% identity in amino acid sequence with each of the other known mammalian CCCs. The corresponding cDNA, obtained from a human heart library and initially termed WO3.3, encodes a 914-residue polypeptide of 96.2 kDa (calculated mass). Sequence analyses predict a 12-transmembrane domain (tm) region, twoN-linked glycosylation sites between tm5 and tm6, and a large intracellular carboxyl terminus containing protein kinase C phosphorylation sites. Northern blot analysis uncovers an ∼3.7-kilobase pair transcript present in muscle, placenta, brain, and kidney. With regard to function, WO3.3 expressed either in HEK-293 cells or Xenopus laevis oocytes does not increase Rb+-, Na+-, and Cl−-coupled transport during 5- or 6-h fluxes, respectively. In the oocyte, however, WO3.3 specifically inhibits human NKCC1-mediated 86Rb+ flux. In addition, coimmunoprecipitation studies using lysates from WO3.3-transfected HEK-293 cells suggest a direct interaction of WO3.3 with endogenous NKCC. Thus, we have cloned and characterized the first putative heterologous CCC-interacting protein (CIP) known at present. CIP1 may be part of a novel family of proteins that modifies the activity or kinetics of CCCs through heterodimer formation.

Homooligomeric and heterooligomeric associations between K+-Cl- cotransporter isoforms and between K+-Cl- and Na+-K+-Cl- cotransporters.

Little is known regarding the quaternary structure of cation-Cl– cotransporters (CCCs) except that the Na+-dependent CCCs can exist as homooligomeric units. Given that each of the CCCs exhibits unique functional properties and that several of these carriers coexist in various cell types, it would be of interest to determine whether the four K+-Cl– cotransporter (KCC) isoforms and their splice variants can also assemble into such units and, more importantly, whether they can form heterooligomers by interacting with each other or with the secretory Na+-K+-Cl– cotransporter (NKCC1). In the present work, we have addressed these questions by conducting two groups of analyses: 1) yeast two-hybrid and pull-down assays in which CCC-derived protein segments were used as both bait and prey and 2) coimmunoprecipitation and functional studies of intact CCCs coexpressed in Xenopus laevis oocytes. Through a combination of such analyses, we have found that KCC2 and KCC4 could adopt various oligomeric states (in the form of KCC2-KCC2, KCC4-KCC4, KCC2-KCC4, and even KCC4-NKCC1 complexes), that their carboxyl termini were probably involved in carrier assembly, and that the KCC4-NKCC1 oligomers, more specifically, could deploy unique functional features. Through additional coimmunoprecipitation studies, we have also found that KCC1 and KCC3 had the potential of assembling into various types of CCC-CCC oligomers as well, although the interactions uncovered were not characterized as extensively, and the protein segments involved were not identified in yeast two-hybrid assays. Taken together, these findings could change our views on how CCCs operate or are regulated in animal cells by suggesting, in particular, that cation-Cl– cotransport achieves higher levels of functional diversity than foreseen.

Increased Immunoreactive Endothelin-1 Levels in Blood Vessels and Glomeruli of Rats with Reduced Renal Mass

Elevated plasma and urinary endothelin-1 (ET-1) levels have been reported in patients with renal failure as well as in remnant kidney models of chronic renal failure. We investigated whether these changes are related to increased ET-1 production in cardiovascular and renal tissues of rats with reduced renal mass. In uremic rats, systolic blood pressure rose in parallel with the progression of renal insufficiency. At week 6, changes in systolic blood pressure were positively correlated with serum creatinine levels (r = 0.728, p < 0.01). Plasma immunoreactive ET-1 (ir-ET-1) concentration was similar in uremic rats and sham-operated controls. In contrast, urinary ir-ET-1 excretion was significantly greater in uremic rats and was correlated with the elevation of serum creatinine and proteinuria (r = 0.795, and 0.922, p < 0.01, respectively). Compared to the controls, ir-ET-1 concentration in the thoracic aorta, preglomerular arteries and glomeruli were 1.4-, 3.5- and 6.7-fold higher, respectively, in uremic rats (p < 0.01) than in the controls. However, ir-ET-1 concentration in the mesenteric arterial bed and the left ventricle remained similar in the 2 groups, whereas it was significantly lower in the renal papilla of uremic rats (p < 0.01). Thus, ET-1 production is unchanged or slightly increased in extrarenal cardiovascular tissues of rats with reduced renal mass. In contrast, ET-1 production is significantly augmented in preglomerular arteries and glomeruli, but reduced in the papilla, suggesting that increased urinary ir-ET-1 excretion in uremic rats reflects ET-1 overproduction in the former renal tissues. Elevated ET-1 production in blood vessels and glomeruli may thus play a key role in the aggravation of hypertension and the progression of renal insufficiency in this rat remnant kidney model of chronic renal failure.

Molecular characterization of a human cation-Cl- cotransporter (SLC12A8A, CCC9A) that promotes polyamine and amino acid transport.

Cation‐Cl− cotransporters (CCCs) belong to a large family of proteins that includes 9 isoforms, two of which have still not been ascribed a transport function (CCC8 and CCC9) while the others are all known to promote Cl−‐coupled Na+ and/or K+ movement at the cell surface. The CCCs are also included in a larger family termed amino acid‐polyamine‐organocation carriers (APCs). In contrast to the CCCs, however, polyamine (PA) transporters have thus far been isolated from unicellular species exclusively and do not all belong to the APC family. In this work, we have found that a splice variant of CCC9 (CCC9a) promotes PA‐amino acid transport at the surface of HEK‐293 cells. We have also found that the influx of PAs in CCC9a‐expressing cells is inhibited by pentamidine as well as furosemide, and that it increases further in the presence of specific amino acids but not of Na+, K+, or Cl−. Hence, a group of substrates that are directly transported by CCC9 and the molecular identity of a PA transport system in animal cells may have been uncovered for the first time. These findings are of special interest given that intracellular PAs play a key role in cell proliferation. J. Cell. Physiol. 220: 680–689, 2009.

Identification of Key Functional Domains in the C Terminus of the K+-Cl– Cotransporters

The K+-Cl– cotransporter (KCC) isoforms constitute a functionally heterogeneous group of ion carriers. Emerging evidence suggests that the C terminus (Ct) of these proteins is important in conveying isoform-specific traits and that it may harbor interacting sites for 4β-phorbol 12-myristate 13-acetate (PMA)-induced effectors. In this study, we have generated KCC2-KCC4 chimeras to identify key functional domains in the Ct of these carriers and single point mutations to determine whether canonical protein kinase C sites underlie KCC2-specific behaviors. Functional characterization of wild-type (wt) and mutant carriers in Xenopus laevis oocytes showed for the first time that the KCCs do not exhibit similar sensitivities to changes in osmolality and that this distinguishing feature as well as differences in transport activity under both hypotonic and isotonic conditions are in part determined by the residue composition of the distal Ct. At the same time, several mutations in this domain and in the proximal Ct of the KCCs were found to generate allosteric-like effects, suggesting that the regions analyzed are important in defining conformational ensembles and that isoform-specific structural configurations could thus account for variant functional traits as well. Characterization of the other mutants in this work showed that KCC2 is not inhibited by PMA through phosphorylation of its canonical protein kinase C sites. Intriguingly, however, the substitutions N728S and S940A were seen to alter the PMA effect paradoxically, suggesting again that allosteric changes in the Ct are important determinants of transport activity and, furthermore, that the structural configuration of this domain can convey specific functional traits by defining the accessibility of cotransporter sites to regulatory intermediates such as PMA-induced effectors.

Novel Insights Regarding the Operational Characteristics and Teleological Purpose of the Renal Na+-K+-Cl2 Cotransporter (NKCC2s) Splice Variants

The absorptive Na+-K+-Cl− cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H2O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H2O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2–NKCC2 interactions.

Inhibition of Na(+)-K(+)-2Cl(-) cotransport by mercury.

Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH(-)) groups. The inorganic form (Hg(2+)) is toxic to epithelial tissues and interacts with various transport proteins including the Na(+) pump and Cl(-) channels. In this study, we determined whether the Na(+)-K(+)-Cl(-) cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues, is also affected by mercurial substrates. To characterize the interaction, we measured the effect of Hg(2+) on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg(2+) inhibits Na(+)-K(+)-Cl(-) cotransport, with inhibitor constant (K(i)) values of 25 microM for the shark carrier (sNKCC1) and 43 microM for the human carrier. In further studies, we took advantage of species differences in Hg(2+) affinity to identify residues involved in the interaction. An analysis of human-shark chimeras and of an sNKCC1 mutant (Cys-697-->Leu) reveals that transmembrane domain 11 plays an essential role in Hg(2+) binding. We also show that modification of additional SH(-) groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.

Aquaporins Mediate Silicon Transport in Humans

In animals, silicon is an abundant and differentially distributed trace element that is believed to play important biological functions. One would thus expect silicon concentrations in body fluids to be regulated by silicon transporters at the surface of many cell types. Curiously, however, and even though they exist in plants and algae, no such transporters have been identified to date in vertebrates. Here, we show for the first time that the human aquaglyceroporins, i.e., AQP3, AQP7, AQP9 and AQP10 can act as silicon transporters in both Xenopus laevis oocytes and HEK-293 cells. In particular, heterologously expressed AQP7, AQP9 and AQP10 are all able to induce robust, saturable, phloretin-sensitive silicon transport activity in the range that was observed for low silicon rice 1 (lsi1), a silicon transporter in plant. Furthermore, we show that the aquaglyceroporins appear as relevant silicon permeation pathways in both mice and humans based on 1) the kinetics of substrate transport, 2) their presence in tissues where silicon is presumed to play key roles and 3) their transcriptional responses to changes in dietary silicon. Taken together, our data provide new evidence that silicon is a potentially important biological element in animals and that its body distribution is regulated. They should open up original areas of investigations aimed at deciphering the true physiological role of silicon in vertebrates.

Phosphoregulation of K+‐Cl− cotransporter 4 during changes in intracellular Cl− and cell volume

It has long been stated that the K+‐Cl− cotransporters (KCCs) are activated during cell swelling through dephosphorylation of their cytoplasmic domains by a protein phosphatase (PP) but that other enzymes are involved by targeting this PP or the KCCs directly. To date, however, the role of signaling intermediates in KCC regulation has been deduced from indirect evidence rather than in vitro phosphorylation studies, and examined after simulation of ion transport through cell swelling or N‐ethylmaleimide treatment. In this study, the oocyte expression system was used to examine the effects of changes in cell volume (CVOL) and intracellular [Cl−] ([Cl−]i) on the activity and phosphorylation levels (PLEV) of KCC4, and determine whether these effects are mediated by PP1 or phorbol myristate acetate (PMA)‐sensitive effectors. We found that (1) low [Cl−]i or low CVOL leads to decreased activity but increased PLEV, (2) high CVOL leads to increased activity but no decrease in PLEV and (3) calyculin A (Cal A) or PMA treatment leads to decreased activity but no increase in PLEV. Thus, we have shown for the first time that one of the KCCs can be regulated through direct phosphorylation, that changes in [Cl−]i or CVOL modify the activity of signaling enzymes at carrier sites, and that the effectors directly involved do not include a Cal A‐sensitive PP in contrast to the widely held view. J. Cell. Physiol. 219: 787–796, 2009.

Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.