A comparative functional analysis of plasma membrane Ca2+ pump isoforms in intact cells.
The four basic isoforms of the plasma membrane Ca2+ pump and the two C-terminally truncated spliced variants PMCA4CII(4a) and 3CII(3a) were transiently overexpressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum, and the mitochondria. As PMCA3CII(3a) had not yet been cloned and studied, it was cloned for this study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously, PMCA3CII(3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca2+ in the native intracellular environment. The level of [Ca2+] in the endoplasmic reticulum and the height of the [Ca2+] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full-length variants in influencing the homeostasis of Ca2+ in the cytosol and the organelles. In particular, PMCA4CII(4a) was as effective as PMCA4CI(4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo.
Brini
Marisa
M
Department of Biochemistry and Center for the Study of Biomembranes of the National Research Council (CNR), University of Padova, Viale G. Colombo 3, Italy. marisa.brini@unipd.it
Coletto
Luisa
L
Pierobon
Nicola
N
Kraev
Natasha
N
Guerini
Danilo
D
Carafoli
Ernesto
E
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D006801:Humans; D053499:Plasma Membrane Calcium-Transporting ATPases
D002147:Calmodulin; D027682:Cation Transport Proteins; D053499:Plasma Membrane Calcium-Transporting ATPases; C506958:ATP2B2 protein, human; C506962:ATP2B3 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium