Abstract
The possibility that certain integral plasma membrane (PM) proteins involved in Ca(2+) 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(+)/Ca(2+) exchanger type 1 (NCX1), a key PM Ca(2+) transporter, was immunoprecipitated from the detergent-soluble fraction. Several abundant PM proteins co-immunoprecipitated with NCX1, including the alpha2 and alpha3 isoforms of the Na(+) pump catalytic (alpha) subunit, and the alpha2 subunit of the dihydropyridine receptor. The adaptor protein, ankyrin 2 (Ank 2), and the cytoskeletal proteins, alpha-fodrin and beta-spectrin, also selectively co-immunoprecipitated with NCX1, as did the ER proteins, Ca(2+) pump type 2 (SERCA 2), and inositol-trisphosphate receptor type 1 (IP(3)R-1). In contrast, a number of other abundant PMs, adaptors, and cytoskeletal proteins did not co-immunoprecipitate with NCX1, including the Na(+) pump alpha1 isoform, PM Ca(2+) pump type 1 (PMCA1), beta-fodrin, and Ank 3. In reciprocal experiments, immunoprecipitation with antibodies to the Na(+) pump alpha2 and alpha3 isoforms, but not alpha1, co-immunoprecipitated NCX1; the antibodies to alpha1 did, however, co-immunoprecipitate PMCA1. Antibodies to Ank 2, alpha-fodrin, beta-spectrin and IP(3)R-1 all co-immunoprecipitated NCX1. Immunocytochemistry revealed partial co-localization of beta-spectrin with NCX1, Na(+) pump alpha3, and IP(3)R-1 in neurons and of alpha-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 alpha2 or alpha3 subunits form Ca(2+) signaling complexes with underlying ER containing SERCA2 and IP(3)R-1. These PM and ER components appear to be linked through the cytoskeletal spectrin network, to which they are probably tethered by Ank 2.
Highlights
Cytosolic Ca2ϩ plays a key role as a second messenger in all cells and is responsible for regulating numerous cellular processes simultaneously
Organization of plasma membrane (PM)-endoplasmic reticulum (ER) Junctions Involved in Ca2ϩ Signaling—This study shows that the PM Naϩ/Ca2ϩ exchanger, Na؉/Ca2؉ exchanger type 1 (NCX1), in neurons and glia, is a component of large, multimolecular complexes that include other PM and ER transporters with which NCX1 is functionally associated
The efficacy of co-IP of these PM proteins, the results of the reciprocal co-IP experiments, and the immunocytochemical evidence of co-localization all demonstrate that both neurons and astrocytes have specialized PMjER complexes
Summary
Preparation of Tissue Extracts—Brains from Sprague-Dawley rats (males, 150 –200 g) were minced in NaCl/sucrose buffer (ϳ1 mg/5 ml) containing 140 mM NaCl, 2 mM EDTA, 10 mM sodium azide, 20 mM Tris base, pH 7.4, 0.25 M sucrose, and protease inhibitor mixture tablets (Roche Diagnostics). Membranes were incubated with secondary antibodies to rabbit IgG (for pAb) or mouse IgG (for primary mAb), conjugated to horseradish peroxidase, for 1 h at room temperature. After blocking nonspecific binding with 10% bovine serum albumin in PBS for 1 h, the neurons were incubated overnight at 4 °C with primary antibodies diluted in Antibody Buffer (0.5 mM NaCl, 10 mM MgCl2, 20 mM NaN3, 20 mM Tris-HCl, pH 7.4). The neurons were washed 3 times with PBS and incubated for 1 h at 22 °C with fluorescently labeled secondary antibodies (Alexa 586-labeled antimouse or anti-rabbit antibodies from Molecular Probes (Eugene, OR) for NCX1, the ␣3 subunit of the Naϩ pump, or IP3R-1, and fluoresceinlabeled donkey anti-chicken antibody for -spectrin). Following washing in Antibody Buffer, the coverslips were mounted with Vectashield
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