Little is known regarding the quaternary structure of cationCl ؊ 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 (1-3), and the KCCs exist as four isoforms and several splice variants (at least five for KCC3 and two for KCC1) (4 -11). All of these structures are predicted to contain 12 transmembrane domains flanked by cytoplasmic termini (1,(12)(13)(14)(15)(16).In mammals, NKCC1 as well as KCC1, -3, and -4 have been shown to exhibit wide tissue distributions, whereas KCC2 is apparently confined to the nervous system (4 -11, 16 -21). They have also been shown to coexist in certain cell types, such as erythrocytes or lens cells, where a number of isoforms/variants (KCC1, KCC3, KCC4) have been identified (10, 11). In certain tissues, localization studies have suggested a more differential distribution (9, 16 -21).Although very homologous to each other, the KCCs display variant affinities for each of the transported ions and for the drug furosemide. In addition, their transport capacity and response to various stimuli are not the same under controlled conditions. In Xenopus laevis oocytes, for example, heterologously expressed KCC2 displays higher K m values for Cl Ϫ but lower K m values for Rb ϩ compared with KCC1 and KCC3 (16,(22)(23)(24)(25). Along the same line, KCC4 is less active than KCC2 at low levels of intracellular Cl Ϫ but more sensitive to phorbol ester-triggered events (26). Not surprisingly, differences between KCCs and NKCCs are even more pronounced (4,16,27).Several lines of evidence suggest that the NKCCs exist as homooligomers in cells. They are as follows. 1) The size of NKCC1 and NKCC2 has been found to increase by a ϳ2-fold factor when membranes expressing either protein were treated with cross-linking agents (12, 28). 2) Through GST pull-down assays and yeast two-hybrid mapping analyses, the cytosolic carboxyl terminus (Ct) of the NKCCs was found to harbor two domains that are endowed with self-interacting properties, * This work was supported by grants from the Kidney Foundation of Canada and from the Canadian Institute of Health and Research (CIHR) through MOP-68949 and MOP-15405. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental

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.

The first isoform of the Na ؉ -K ؉ -Cl ؊ cotransporter (NKCC1), a widely distributed member of the cation-Cl ؊ cotransporter superfamily, plays key roles in many physiological processes by regulating the ion and water content of animal cells and by sustaining electrolyte secretion across various epithelia. Indirect studies have led to the prediction that NKCC1 operates as a dimer assembled through binding domains that are distal to the amino portion of the carrier. In this study, evidence is presented that NKCC1 possesses self-interacting properties that result in the formation of a large complex between the proximal and the distal segment of the cytosolic C terminus. Elaborate mapping studies of these segments showed that the contact sites are dispersed along the entire C terminus, and they also led to the identification of a critical interacting residue that belongs to a putative forkhead-associated binding domain. In conjunction with previous findings, our results indicate that the uncovered interacting domains are probably a major determinant of the NKCC1 conformational landscape and assembly into a high order structure. A model is proposed in which the carrier could alternate between monomeric and homo-oligomeric units via chemical-or ligand-dependent changes in conformational dynamics.

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.