Exon loss accounts for differential sorting of Na-K-Cl cotransporters in polarized epithelial cells

Abstract

The renal Na-K-Cl cotransporter (NKCC2) is selectively expressed in the apical membranes of cells of the mammalian kidney, where it is the target of the clinically important loop diuretics. In contrast, the "secretory" NKCC1 cotransporter is localized in the basolateral membranes of many epithelia. To identify the sorting signal(s) that direct trafficking of NKCCs, we generated chimeras between the two isoforms and expressed these constructs in polarized renal epithelial cell lines. This analysis revealed an amino acid stretch in NKCC2 containing apical sorting information. The NKCC1 C terminus contains a dileucine motif that constitutes the smallest essential component of its basolateral sorting signal. NKCC1 lacking this motif behaves as an apical protein. Examination of the NKCC gene structure reveals that this dileucine motif is encoded by an additional exon in NKCC1 absent in NKCC2. Phylogenetic analysis of this exon suggests that the evolutionary loss of this exon from the gene encoding the basolateral NKCC1 constitutes a novel mechanism that accounts for the apical sorting of the protein encoded by the NKCC2 gene.

Figure 1.

Localization of NKCC1, NKCC2 and chimeras in transiently transfected MDCK cells. The chimeras were generated by exchanging the three major portions of the contransporter: the N terminus, TM, and the C terminus. The structure of each chimera is shown in the inset of the en face image in which its localization is depicted. The NKCC1 regions are shown in gray, and the NKCC2 regions are shown in black. The sorting information is contained within the C termini of NKCC proteins.

Figure 2.

Localizations of sequential C terminus chimeras. Confocal xz cross-section immunofluorescence images are presented. Immunofluorescence analysis was performed on stably transfected MDCK cells. The junction point of each chimera is shown on the left of the panel in which its localization is depicted (numbering in replaced human NKCC1, NKCC1 C terminus in gray, NKCC2 C terminus in black). The sorting information is embedded in the amino acid region between the junction points of chimeras IV and V.

Figure 3.

(A) Cell surface distribution of the sequential chimeras determined by domain-selective biotinylation in stably transfected MDCK cells. (B) Rate of ⁸⁶Rb⁺ influx mediated by chimeras I, III, IV, V, and NKCC1 in stably transfected HEK cells after preincubation in various media. The data are relative to the maximal activation of NKCC1 and are corrected for NKCC protein expression. Data are presented as the range of two measurements in a representative experiment. All chimeras are functionally active.

Figure 4.

Localization of PLAP-V constructs and chimera IV-V. (A) The structure of each PLAP-V construct is shown on the left of the panel in which its localization is depicted. The IV-V region of NKCC2 carries autonomous apical sorting information. (B) The structure of the chimera IV-V is shown on the left of the panel in which its localization is depicted. The NKCC1 portions are shown in gray and the NKCC2 portions in black. The IV-V NKCC2 region is not, in itself, sufficient to induce the fully apical localization of NKCC1.

Figure 5.

(A) Immunolocalization of chimeras IV-VI, V-VI, IV-V+V-term, III-V in stably transfected MDCK cells. xz cross-section immunofluorescence images are presented. The structure of each chimera is shown on the right of the panel in which its localization is depicted. The NKCC1 portions are shown in gray and the NKCC2 portions in black. The NKCC2 sequence downstream of the IV-V NKCC2 region is necessary for apical sorting. (B) Domain selective cell surface biotinylation of chimeras IV-V and chimera IV-VI (A, apical; B, basolateral). (C) Rate of ⁸⁶Rb⁺ influx mediated by chimeras IV-V, IV-VI, and NKCC1 in stably transfected HEK cells. The data are relative to the maximal activation of NKCC1 and are shown as average of three flux rows of a representative experiment. Chimeras IV-V and IV-VI are functionally identical to wild-type NKCC1.

Figure 6.

Detergent solubility assay of the apical chimera III and the basolateral chimera V. MDCK cells stably expressing chimera III and chimera V were subjected to the detergent solubility assay as described in Materials and Methods. A representative experiment is shown. Neither chimera is associated with detergent-insoluble membrane domains.

Figure 7.

Localization of NKCC1ΔA, NKCC1ΔB, NKCC1-LL/AA, and NKCC1-EEED/QQQN mutants in stably transfected MDCK cells. (A and B) Deleted regions in NKCC1 are represented by dotted lines in the cartoons. Basolateral sorting information is contained in the ΔB region of NKCC1. (C) NKCC1-LL/AA mutant was localized on the apical membrane suggesting that this dileucine motif is the smallest component of the basolateral NKCC1 sorting signal. (D) NKCC1-EEED/QQQN mutant was localized on the basolateral membrane, suggesting that the EEED motif is not involved in the basolateral sorting. (E) Domain-selective cell surface biotinylation of NKCC1ΔA, NKCC1ΔB, NKCC1-LL/AA, and NKCC1-EEED/QQQN mutants.

Figure 8.

Cell surface localization of PLAP-V-NKCC1 constructs and chimera III-IV+VI-term. The structure of each construct is shown on the left of the panel in which its localization is depicted. (A) The IV-V region of NKCC1 carries autonomous basolateral sorting signals. (B) The dileucine is the critical motif in the NKCC1 IV-V sorting region. (C) The IV-V region of NKCC1 is sufficient to confer a fully basolateral localization to an apical chimera.

Figure 9.

Exon structure of NKCCs. (A) Alignment of NKCC1 and NKCC2 sequences in the exon 20–21 region, color-coded by similarity to human NKCC1. (B) Exon organization of NKCC1 and NKCC2, with every other exon highlighted in light or dark blue. Exon 21, unique to NKCC1, is shown in red.