Plasma membrane-cytoskeleton-endoplasmic reticulum complexes in neurons and astrocytes.
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.
Lencesova
Lubomira
L
Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
O'Neill
Andrea
A
Resneck
Wendy G
WG
Bloch
Robert J
RJ
Blaustein
Mordecai P
MP
D000818:Animals; D017487:Ankyrins / Q000378:metabolism; D001253:Astrocytes / Q000378:metabolism* / Q000648:ultrastructure; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D013049:Spectrin / Q000378:metabolism
D017487:Ankyrins; D008565:Membrane Proteins; D013049:Spectrin