Plasma membrane calcium pump activity is affected by the membrane protein concentration: evidence for the involvement of the actin cytoskeleton.
Plasma membrane calcium pumps (PMCAs) are integral membrane proteins that actively expel Ca(2+) from the cell. Specific Ca(2+)-ATPase activity of erythrocyte membranes increased steeply up to 1.5-5 times when the membrane protein concentration decreased from 50 microg/ml to 1 microg/ml. The activation by dilution was also observed for ATP-dependent Ca(2+) uptake into vesicles from Sf9 cells over-expressing the PMCA 4b isoform, confirming that it is a property of the PMCA. Dilution of the protein did not modify the activation by ATP, Ca(2+) or Ca(2+)-calmodulin. Treatment with non-ionic detergents did not abolish the dilution effect, suggesting that it was not due to resealing of the membrane vesicles. Pre-incubation of erythrocyte membranes with Cytochalasin D under conditions that promote actin polymerization abolished the dilution effect. Highly-purified, micellar PMCA showed no dilution effect and was not affected by Cytochalasin D. Taken together, these results suggest that the concentration-dependent behavior of the PMCA activity was due to interactions with cytoskeletal proteins. The dilution effect was also observed with different PMCA isoforms, indicating that this is a general phenomenon for all PMCAs.
Vanagas
Laura
L
IQUIFIB, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.
Rossi
Rolando C
RC
Caride
Ariel J
AJ
Filoteo
Adelaida G
AG
Strehler
Emanuel E
EE
Rossi
Juan Pablo F C
JP
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D015638:Cytochalasin D; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000166:cytology; D006801:Humans; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000378:metabolism*; D018411:Spodoptera
D015638:Cytochalasin D; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium