Immunologic localization and kinetic characterization of a Na +/Ca 2+ exchanger in neuronal and non-neuronal cells

Abstract

The plasma membrane Na +/Ca 2+ exchanger is believed to play a role in the regulation of Ca 2+ fluxes in neurons, though the lack of specific inhibitors has limited the delineation of its precise contribution. We recently reported the development of antibodies against a 36-kDa brain synaptic membrane protein which immunoprecipitated exchanger activity from solubilized membranes. In the present study we examined the kinetics of the Na +/Ca 2+ exchanger in primary neurons in culture, in a neuronal hybrid cell line (NCB-20), and in a fibroblast-like cell line (CV-1) to see whether the level of exchanger activity correlated with the degree of immunostaining produced by our antibodies. The V max was determined for each cell type and found to be highest in primary neurons. Exchanger activity increased in primary neurons between days 1 and 6 in culture, but no such time-dependent change occurred in either of the cell lines. Immunoblot analysis of the three cell types probed with the anti-36-kDa protein antibodies revealed significantly greater immunostaining in the primary neurons compared with the other two cell types. Intensity of staining of neurons also increased significantly between days 1 and 6 in culture. Immunocytochemistry showed significant labelling of the primary neurons on the neuritic processes and points of contact between cells. The NCB-20 and CV-1 cells showed considerably lower levels of immunoreactivity. The antibodies immunoextracted ∼90% of the exchanger activity in the primary neurons and ∼70 and 50% of the activity in NCB-20 and CV-1 cells respectively. Thus the expression of the 36-kDa protein appears to be closely associated with the Na +/Ca 2+ exchanger in neuronal cells and, possibly to a lesser extent, in non-neuronal cells.