Phospholamban regulates the Ca2+-ATPase through intramembrane interactions.
There is clear evidence for direct regulatory protein-protein interactions between phospholamban (PLN) and the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a) in cytoplasmic domains, but there is less clear evidence for regulatory interactions in the transmembrane domains of the two proteins. We have now coexpressed SERCA isoforms with the transmembrane sequence of PLN and with epitope-tagged transmembrane sequences of PLN to study intramembrane interactions in the absence of cytoplasmic interactions. Coexpression of the transmembrane sequence of phospholamban (Met-PLN28-52) with SERCA1a, SERCA2a, and SERCA3 inhibited Ca2+ transport by lowering apparent Ca2+ affinity. Addition of the hemagglutinin (HA) epitope to the transmembrane sequence of PLN (HA-PLN28-52) or deletion of PLN residues 21-29 (PLN1-20-PLN30-52) "supershifted" apparent Ca2+ affinity to values lower than those observed with native PLN without uncoupling Ca2+ transport from ATP hydrolysis. Inhibition by PLN1-20-PLN30-52 or by Flag-PLN28-52 was reversed by PLN antibody or by Flag antibody, demonstrating that inhibition by these constructs is reversible and that the inhibitory constructs are properly oriented in the membrane. These results suggest that PLN modulates the apparent Ca2+ affinity of SERCA2a through intramembrane interactions, which are disrupted at long range and in concert with disruption of the well characterized cytoplasmic interactions.
Kimura
Y
Y
Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, Toronto, Ontario M5G 1L6, Canada.
Kurzydlowski
K
K
Tada
M
M
MacLennan
D H
DH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014162:Transfection; D000099211:Phospholamban
D000255:Adenosine Triphosphate; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D018076:DNA, Complementary; D000099211:Phospholamban