Immunocytochemical localization of the plasma membrane calcium pump, calbindin-D28k, and parvalbumin in Purkinje cells of avian and mammalian cerebellum.
A monoclonal antibody produced against the human erythrocyte plasma membrane calcium pump (PMCA) was shown to react immunohistochemically with an epitope of the PMCA in avian and mammalian cerebellum. Western blot analysis of purified synaptosomes and homogenates from avian cerebellum revealed major immunoreactive proteins with molecular masses (130 kDa and 138 kDa) similar to those of purified erythrocyte PMCA. Dual-imaging confocal immunofluorescence microscopy of avian cerebellum showed that the PMCA antibody stained the periphery of the soma whereas calbindin-D28k was located in the cytosol. PMCA heavily stained the more distal dendrites of the Purkinje cells and, within the resolution of the fluorescence procedure, colocalized with calbindin-D28k. By using alkaline phosphatase-conjugated second antibody, PMCA was again localized to the peripheral soma, to a segmental pattern in dendrites, and to presumed spiny elements. The soma periphery and dendrites of Purkinje cells of the rat cerebellum were also prominently stained with anti-PMCA antibody and compared to parvalbumin localization. Dendritic depolarization and dendritic spiking behavior are significant Ca(2+)-dependent events of Purkinje cells. The rapid decline of intracellular free Ca2+ after the rapid rise time of Ca2+ transients is considered to be due to sequestration by Ca2+ buffers, uptake by intracellular stores, and Ca2+ extrusion mechanisms, the latter a function of PMCA now shown immunohistochemically to be a prominent feature of Purkinje cell dendrites.
Tolosa de Talamoni
N
N
Department of Physiology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.
Smith
C A
CA
Wasserman
R H
RH
Beltramino
C
C
Fullmer
C S
CS
Penniston
J T
JT
D000469:Alkaline Phosphatase; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D064092:Calbindin 1; D064026:Calbindins; D000252:Calcium-Transporting ATPases / Q000032:analysis*; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002531:Cerebellum / Q000166:cytology*; D002645:Chickens; D007150:Immunohistochemistry / Q000379:methods; D008856:Microscopy, Fluorescence; D010320:Parvalbumins / Q000032:analysis*; D011689:Purkinje Cells / Q000166:cytology* / Q000201:enzymology; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000032:analysis*; D013574:Synaptosomes / Q000737:chemistry / Q000648:ultrastructure
D000906:Antibodies; C576498:CALB1 protein, human; C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D010320:Parvalbumins; D064030:S100 Calcium Binding Protein G; D000469:Alkaline Phosphatase; D000252:Calcium-Transporting ATPases