Sodium-calcium exchanger complexed with GM1 ganglioside in nuclear membrane transfers calcium from nucleoplasm to endoplasmic reticulum

Abstract

The inner membrane of the nuclear envelope (NE) was previously shown to contain a Na/Ca exchanger (NCX) tightly linked to GM1 ganglioside that mediates transfer of nucleoplasmic Ca(2+) to the NE lumen and constitutes a cytoprotective mechanism. This transfer was initially observed with isolated nuclei and is now demonstrated in living cells in relation to subcellular Ca(2+) dynamics. Four cell lines with varying expression of NCX and GM1 in the NE were transfected with cameleon-fluorescent Ca(2+) indicators genetically targeted to NE/endoplasmic reticulum (ER) and nucleoplasm to monitor [Ca(2+)](ne/er) and [Ca(2+)](n) respectively. Cytosolic Ca(2+) ([Ca(2+)](cyt)) was indicated with fura-2. Thapsigargin caused progressive loss of [Ca(2+)](ne/er), which was rapidly replaced on addition of extrinsic Ca(2+) to those cells containing fully functional NCX/GM1: differentiated NG108-15 and C6 cells. Reduced elevation of [Ca(2+)](ne/er) following thapsigargin depletion occurred in cells containing little or no GM1 in the NE: undifferentiated NG108-15 and NG-CR72 cells. No change in [Ca(2+)](ne/er) due to applied Ca(2+) was seen in Jurkat cells, which entirely lack NCX. Ca(2+) entry to NE/ER was also blocked by KB-R7943, inhibitor of NCX. [Ca(2+)](n) and [Ca(2+)](cyt) were elevated independent of [Ca(2+)](ne/er) and remained in approximate equilibrium with each other. Ca(2+) rise in the ER originated in the NE region and extended to the entire ER network. These results indicate the nuclear NCX/GM1 complex acts to gate Ca(2+) transfer from cytosol to ER, an alternate route to the sarcoplasmic/endoplasmic reticulum calcium ATPase pump. They also suggest a possible contributory mechanism for independent regulation of nuclear Ca(2+).

Fig. 1.

Expression of NCX and GM1. (A) Nuclei of NG108–15 (differentiated), NG-CR72, C6, and Jurkat cells were isolated, and fixed. GM1 and NCX in the NE were revealed by double staining with CtxB-FITC and anti-NCX Ab (second Ab-linked to Texas red). Images show coexpression of NCX and GM1 in the NE of NG108–15 and C6 cells; NG-CR72 cells showed NCX but no GM1 while Jurkat cells showed GM1 but no NCX. (B) NCX in NE and non-nuclear membrane mixture (M. Mix.) was immunoprecipitated with mouse anti-NCX mAb (C2C12), subjected to SDS/PAGE and electrophoretic transfer to PVDF; it was then blotted with rabbit polyclonal anti-NCX Ab (a). NG108–15 and NG-CR72 cells gave bands corresponding to known NCX pattern in the NE and non-nuclear M. Mix. (e.g., plasma membrane) while C6 cells expressed NCX only in the NE. Jurkat cells were devoid of NCX in both fractions. Parallel blot using CtxB-HRP (b) revealed tight association of GM1 with major NCX bands in the NE of NG108–15 and C6 cells, but not GM1-deficient NG-CR72 cells or NCX-deficient Jurkat cells.

Fig. 2.

Targeted expression of cameleon indicators in NE/ER and nucleoplasm. (A) NG108–15 cells and (B) Jurkat cells were transfected with ER- or Nu-cameleon. ER-cameleon-expressing cells were immunostained with antinuclear pore protein (NPP) Ab and second antibody with Texas red, showing expression of cameleon protein in the NE and throughout the ER. Nu-cameleon-expressing cells were fixed and stained with nuclear-specific dye Hoechst 33342, showing restricted expression in the nucleoplasm.

Fig. 3.

Coordinated Ca²⁺ changes in NE/ER ([Ca²⁺]_ne/er), nucleoplasm ([Ca²⁺]_n), and cytosol ([Ca²⁺]_cyt) in NG108–15 (A) and NG-CR72 (B) cells. NG108–15 cells were differentiated with db-cAMP/KCl, except (Ab) in which undifferentiated NG108–15 cells were used. NG-CR72 cells were differentiated with db-cAMP alone. Cells were transfected with ER- (A a and b and B a and b) or Nu- (Ac and Bc) cameleon, or loaded with fura-2 (Ad and Bd). Ca²⁺ imaging was started in Ca²⁺-free buffer and monitored as R_535/480 for cameleon and R_350/380 for fura-2. Reagents included Tg, NCX blocker KB-R7943 (KB), LIGA-20, and CaCl₂/KCl or CaCl₂/NaCl that were applied as indicated. “Control” in all panels represents CaCl₂/KCl without KB or LIGA-20. LIGA-20, when used, was used for prior bath incubation of cells for 30 min. Each trace was average of >30 cells from 5 to 8 independent runs.

Fig. 4.

Ratio images of [Ca²⁺]_ne/er in differentiated NG108–15 cells. Serial images were obtained with ER-cameleon-expressing cells as described in Fig. 3. (A) Control (no KB-R7943): Following Tg application at 50 s leading to depletion of [Ca²⁺]_ne/er, addition of CaCl₂/KCl at 550 sec caused elevation of [Ca²⁺]_ne/er that originated in the perinuclear region (arrowheads at 592.8 s image with *) and then extended to entire NE/ER. This was followed by gradual Tg-induced depletion. (B) Repeat of above experiment with KB-R7943 showed limited uptake of Ca²⁺ in the perinuclear region at 592.9s (*) consistent with incomplete blockade shown in Fig. 3 Aa and Ba; modest rise in [Ca²⁺]_ne/eroccurred briefly to 637.7 s followed by gradual Tg-induced depletion.

Fig. 5.

Coordinated Ca²⁺ changes in NE/ER, nucleoplasm, and cytosol of C6 and Jurkat cells. C6 (A) and Jurkat (B) cells were transfected with ER- (a) or Nu- (b) cameleon, or loaded with fura2 (c). Ratiometric imaging was carried out as in Fig. 3. Arrows marked with Ca at 550 s represent addition of CaCl₂/KCl mixture for C6 cells or CaCl₂ alone for Jurkat cells. [Ca²⁺]_ne/er was elevated in C6 cells, which express nuclear NCX, but not in Jurkat cells, which lack nuclear NCX. Each trace was average of >30 cells from 5 to 8 independent runs.