Homooligomeric and Heterooligomeric Associations between K+-Cl– Cotransporter Isoforms and between K+-Cl– and Na+-K+-Cl– Cotransporters

Abstract

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

Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1)

“…This finding is consistent with numerous lines of end-point evidence supporting homomultimerization of cation-chloride cotransporters including NKCC1, NKCC2, Na-Cl cotransporter, and KCCs (1,5,(7)(8)(9)(10)(11)(12)(13). The relatively high ratio of intermolecular to intramolecular FRET (Fig.…”

“…Previous observations include dominant-negative behavior of inactive mutants (1,5,(7)(8)(9), cross-linking studies (9,10), yeast two-hybrid experiments (11), and immunoprecipitation experiments (8,9,12,13). KCC2 dimers are apparently stabilized by a disulfide bridge (12), but this does not appear to occur in NKCCs.…”

Differences in the Large Extracellular Loop between the K+-Cl− Cotransporters KCC2 and KCC4

“…The transporters probably become functional by forming oligomers. Coexpression of a truncated transport-inactive KCC1 inhibited transport activity of other KCCs (16), and biochemical analysis demonstrated oligomeric forms of KCCs (17)(18)(19)(20). A similar organization of the transporters is supported by identical functional consequences of mutations.…”

Direct control of Na+-K+-2Cl−-cotransport protein (NKCC1) expression with aldosterone

“…For instance, in response to reductions in intracellular chloride concentrations, Ste20-related proline-alanine-rich kinase (SPAK) phosphorylates NKCC1 to elevate cotransporter activity and raise chloride influx (12,53). Oxidative stress response kinase 1 (OSR1) also phosphorylates and activates NKCC1 in the presence of oxidative stress (51). Therefore, because of its important physiological functions, mis-regulation or deficiencies in the expression or distribution of NKCC1 isoforms in kidney epithelial cells can have negative physiological consequences.…”