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

Abstract

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.

Dietary salt intake modulates differential splicing of the Na-K-2Cl cotransporter NKCC2

“…Similar results were recently obtained for rats. Again, water loading led to decreased expression levels of the NKCC2 A and F isoform (1). In contrast to NKCC2 mRNA expression, vasopressin has been shown to increase NKCC2 protein abundance and the protein phosphorylation status, resulting in augmented membrane trafficking (2,6,9,18,31).…”

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

“…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).…”

“…The NKCCs exist as two isoforms (1)(2)(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).…”

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

“…Because the junction point of this chimera was similar to the one that was used for KCC2 4-2-2 , residues proximal to amino acid 644 could thus play a structural role as well (2). We have recently found that the proximal and distal Ct of the NKCCs, which are closely related to the KCC isoforms, are capable of self-interactions (43,49), suggesting that residues between these distant protein segments can associate with one another in the context of intramolecular and intermolecular interactions.…”

“…Immunolocalization of KCCs Expressed in X. laevis Oocytes-These studies were performed as described in previous publications by our group (41)(42)(43)(44). Briefly, oocytes cryosections (10 m) were postfixed for 30 min in paraformaldehyde and incubated sequentially with a primary and secondary antibody (for 1 h each at room temperature).…”