Plasma membrane calcium pump (PMCA) isoform 4 is targeted to the apical membrane by the w-splice insert from PMCA2

Abstract

Local Ca(2+) signaling requires proper targeting of the Ca(2+) signaling toolkit to specific cellular locales. Different isoforms of the plasma membrane Ca(2+) pump (PMCA) are responsible for Ca(2+) extrusion at the apical and basolateral membrane of polarized epithelial cells, but the mechanisms and signals for differential targeting of the PMCAs are not well understood. Recent work demonstrated that the alternatively spliced w-insert in PMCA2 directs this pump to the apical membrane. We now show that inserting the w-insert into the corresponding location of the PMCA4 isoform confers apical targeting to this normally basolateral pump. Mutation of a di-leucine motif in the C-tail thought to be important for basolateral targeting did not enhance apical localization of the chimeric PMCA4(2w)/b. In contrast, replacing the C-terminal Val residue by Leu to optimize the PDZ ligand site for interaction with the scaffolding protein NHERF2 enhanced the apical localization of PMCA4(2w)/b, but not of PMCA4x/b. Functional studies showed that both apical PMCA4(2w)/b and basolateral PMCA4x/b handled ATP-induced Ca(2+) signals with similar kinetics, suggesting that isoform-specific functional characteristics are retained irrespective of membrane targeting. Our results demonstrate that the alternatively spliced w-insert provides autonomous apical targeting information in the PMCA without altering its functional characteristics.

Fig. 1

PMCA constructs and mutants studied in this work. (A) A scheme of the PMCA with its 10 membrane-spanning segments (numbered 1–10) is shown on the top. The alternative splice site A is indicated, and the x- and w-splice inserts are shown as boxes corresponding to the included exon(s). The position of a di-leucine (LL) motif in the C-tail is also indicated, and the PDZ-binding sequence (PDZ-BD) is represented by a black box. The amino acid sequences corresponding to the A-splice site in PMCA4x, PMCA2x, PMCA4(2w) and PMCA2w, as well as partial sequences of the C-tail including the di-leucine and PDZ-binding motifs of PMCA4x/b, PMCA2x/b, PMCA4x/b-V>L, PMCA4(2w)/b-V>L, and PMCA4(2w)/b-LL>AA are shown below. Amino acid residue numbers are given for the first and last residue in each sequence. The A-splice insert, the di-leucine motif, and the C-terminal residue are in bold print. (B) Expression of the recombinant GFP-tagged PMCA4 constructs. Aliquots of lysate from HeLa cells transfected with the constructs indicated on top of each lane were separated by SDS-PAGE and probed by Western blotting with an anti-GFP antibody to detect the recombinant GFP-PMCA protein (top panel) or with an antibody against GAPDH as loading control (bottom panel). All GFP-PMCA constructs are expressed at comparable levels as full-length proteins of the expected size.

Fig. 2

The 2w-insert confers apical targeting to PMCA4b in polarized MDCK cells. MDCK cells grown for 72 h on Lab-Tek II Chambered Coverglass were transfected with cDNAs encoding the indicated GFP-tagged PMCA variants. 48 h post-transfection, cells were fixed and stained with the apical marker anti-ezrin (indicated in red) and the basolateral marker anti-Na,KATPase (indicated in blue), and analyzed by confocal microscopy. Co-localization of the PMCA (detected by its GFP fluorescence) with ezrin is indicated in yellow in the merged images. Representative apical and middle (lateral) x-y scans are shown in the first and second rows of each panel. In the third row of each panel, x-z projections of the x-y scans are presented. Note the prominent apical localization of the chimeric PMCA4(2w)/b, while PMCA4x/b remains strictly basolateral. Data shown are representative of at least three independent experiments. Scale bars represent 10 μm.

Fig. 3

NHERF2 enhances the apical localization of PMCA4(2w)/b with a C-terminal Val to Leu mutation. MDCK cells were transiently co-transfected with NHERF2 and GFP-PMCA4(2w)/b-V>L or GFP-PMCA4x/b-V>L as indicated. Fixed cells were stained for NHERF2 (red) and Na,K-ATPase (blue) and observed by confocal fluorescence microscopy as described in the legend for Fig. 2. The expressed PMCAs were detected by the GFP fluorescence of their tag. NHERF2 localized to the apical membrane where it markedly co-localized with PMCA4(2w)b-V>L (left panels) but was clearly separated from PMCA4x/b-V>L (right panels), which remained strictly basolateral. Scale bar = 10 μm.

Fig. 4

Quantitative evaluation of the localization of PMCA constructs in MDCK cells and effect of NHERF2 on the apical/basolateral distribution of the PMCAs. MDCK cells were transfected with the indicated constructs and processed as described in the legend for Fig. 2. Localization of the GFP-tagged PMCAs was evaluated by their co-localization with the apical marker ezrin and the basolateral marker Na,K-ATPase. Quantification was performed as described in section 2.4. (A) Ratio of apical to basal/lateral distribution of different PMCA variants in the absence and presence of NHERF2. PMCA4x/b, 4x/b-LL>AA and 4x/b-V>L are strictly basolateral and insensitive to the presence of NHERF2. The 2w splice insert promotes apical localization of PMCA4(2w)/b, 4(2w)/b-LL>AA and 4(2w)/b-V>L. Note the significantly enhanced apical to basolateral ratio for PMCA4(2w)/b-V>L comparable to that of PMCA2w/b. A C-terminal leucine residue permits protein-protein interactions between the PMCA and NHERF2. Therefore, over-expression of NHERF2 further enhances the apical localization of PMCA2w/b and PMCA4(2w)/b-V>L, but not of PMCA4(2w)/b without the C-terminal leucine residue. Also note that mutation of a di-leucine putative basolateral targeting sequence did not enhance apical localization of PMCA4(2w)/b-LL>AA. Values represent the mean ± SEM of calculations from 25 to 50 cells of three independent experiments. Significance by t-test: **p < 0.002; ***p < 0.0001. (B) Summary of the localization of different PMCA constructs in polarized MDCK cells. The 2w-insert functions as primary apical targeting signal; NHERF2 can enhance the apical distribution of the PMCA but only if the pump is targeted to the apical membrane (via the 2w insert) AND contains an optimal NHERF2-interacting C-terminal sequence.

Fig. 5

PMCAs handle global Ca²⁺-signals in an isoform-specific manner regardless of their localization. (A) MDCK cells were cultured on Nunc Lab-Tek Chambered Coverglass and co-transfected with the indicated PMCA construct and the genetically encoded Ca²⁺ indicator GCaMP2. Single-cell Ca²⁺ signal measurements were carried out in Ca²⁺ free HBSS. Ca²⁺ signals were elicited by addition of 100 μM ATP (arrows). 20–30 cells were measured in a single experiment, time-lapse sequences were recorded, images were analyzed and the experimental data were fitted as described in section 2.5. Left panel: average fluorescence profile of Ca²⁺ signals in cells expressing PMCA4(2w)/b (black) and PMCA4×/b (grey). Right panel: average fluorescence profile of Ca²⁺ signals in cells expressing PMCA2w/b (black) and PMCA2×/b (grey). Stippled lines indicate the standard deviation (SD) of each average curve. (B) Quantification of Ca²⁺ signal measurements shown in (A). Data were obtained by calculating the area under the curves of Ca²⁺ signal time-lapse sequences of 40–50 cells from three independent determinations. Data are expressed as mean ± standard error (SE). There was no significant difference in Ca²⁺ handling by the x- and w-variants of either PMCA2 or PMCA4, however, note the distinct difference in Ca²⁺ handling between the slow PMCA4 and the fast PMCA2 isoforms. (C) Confocal images of representative populations of MDCK cells expressing the various PMCA protein variants applying the same detector gain, zoom, amplitude offset and gain, transmission percent, pinhole size, and scan speed for each image. The data indicate that the constructs were equally expressed in MDCK cells.