Identification of a GM1/Sodium–Calcium exchanger complex in the nuclear envelope of non‐neuronal cells

Abstract

Our previous studies identified a Na–Ca exchanger (NCX) that is tightly associated with GM1 ganglioside and potentiated by it in the nuclear envelope (NE) of NG108‐15 cells and primary neurons. The purpose of the present study was to explore whether this is a general phenomena or limited to neurons. Non‐neuronal C6 (glioma), HeLa (Epithelial carcinoma) and NCTC (connective tissue) cell lines were used. Immunocytochemical staining with anti‐NCX antibody and cholera toxin B subunit revealed that NCX and GM1 coexist in the nuclei from all 3 cell lines; in relation to plasma membrane, only HeLa cells showed staining for both NCX and GM1. Purified NE and non‐nuclear membrane mixture (mainly plasma membrane) from the 3 cell lines were immunoprecipitated with a mouse monoclonal anti‐NCX antibody and the precipitated proteins separated on SDS–PAGE. Analysis by immunoblot, showed that NCX is tightly associated with GM1 in the NE of all 3 cell lines. In contrast, NCX and the more loosely associated GM1 from plasma membrane of HeLa cells were separated by SDS–PAGE. Isolated nuclei from C6 cells were used for 45Ca2+ uptake experiments, which provided functional evidence that this exchanger protein is strongly potentiated by GM1. In similar experiments with Jurkat cells (T lymphocyte), no NCX was found. These results suggest a possible new and widely distributed mechanism for regulation of nuclear calcium by NCX in association with GM1.Acknowledgements: supported by NIH grant NS 33912.