Plasma Membrane-Cytoskeleton-Endoplasmic Reticulum Complexes in Neurons and Astrocytes

Abstract

The possibility that certain integral plasma membrane (PM) proteins involved in Ca2+ homeostasis form junctional units with adjacent endoplasmic reticulum (ER) in neurons and glia was explored using immunoprecipitation and immunocytochemistry. Rat brain membranes were solubilized with the mild, non-ionic detergent, IGEPAL CA-630. Na +/Ca2+ exchanger type 1 (NCX1), a key PM Ca2+ transporter, was immunoprecipitated from the detergent-soluble fraction. Several abundant PM proteins co-immunoprecipitated with NCX1, including the α2 and α3 isoforms of the Na+ pump catalytic (α) subunit, and the α2 subunit of the dihydropyridine receptor. The adaptor protein, ankyrin 2 (Ank 2), and the cytoskeletal proteins, α-fodrin and β-spectrin, also selectively co-immunoprecipitated with NCX1, as did the ER proteins, Ca2+ pump type 2 (SERCA 2), and inositol-trisphosphate receptor type 1 (IP3R-1). In contrast, a number of other abundant PMs, adaptors, and cytoskeletal proteins did not co-immunoprecipitate with NCX1, including the Na+ pump α1 isoform, PM Ca2+ pump type 1 (PMCA1), β-fodrin, and Ank 3. In reciprocal experiments, immunoprecipitation with antibodies to the Na+ pump α2 and α3 isoforms, but not α1, co-immunoprecipitated NCX1; the antibodies to α1 did, however, co-immunoprecipitate PMCA1. Antibodies to Ank 2, α-fodrin, β-spectrin and IP3R-1 all co-immunoprecipitated NCX1. Immunocytochemistry revealed partial co-localization of β-spectrin with NCX1, Na+ pump α3, and IP3R-1 in neurons and of α-fodrin with NCX1 and SERCA2 in astrocytes. The data support the idea that in neurons and glia PM microdomains containing NCX1 and Na+ pumps with α2 or α3 subunits form Ca2+ signaling complexes with underlying ER containing SERCA2 and IP3R-1. These PM and ER components appear to be linked through the cytoskeletal spectrin network, to which they are probably tethered by Ank 2.