PHOSPHOLAMBAN REGULATES THE CA2-ATPASE THROUGH INTRAMEMBRANE INTERACTIONS()

There is clear evidence for direct regulatory protein-protein interact ions between phospholamban (PLN) and the Ca2+-ATPase of cardiac sarcop lasmic reticulum (SERCA2a) in cytoplasmic domains, but there is less c lear 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 se quences of PLN to study intramembrane interactions in the absence of c ytoplasmic interactions. Coexpression of the transmembrane sequence of phospholamban (Met-PLN(28-52)) with SERCA1a, SERCA2a, and SERCA3 inhi bited Ca2+ transport by lowering apparent Ca2+ affinity. Addition of t he hemagglutinin (IIA) epitope to the transmembrane sequence of PLN (H A-PLN(28-52)) or deletion of PLN residues 21-29 (PLN(1-20)-PLN(30-52)) ''supershifted'' apparent Ca2+ affinity to values lower than those ob served with native PLN without uncoupling Ca2+ transport from ATP hydr olysis. Inhibition by PLN(1-20)-PLN(30-52) or by Flag-PLN(28-52) was r eversed by PLN antibody or by Flag antibody, demonstrating that inhibi tion by these constructs is reversible and that the inhibitory constru cts are properly oriented in the membrane. These results suggest that PLN modulates the apparent Ca2+ affinity of SERCA2a through intramembr ane interactions, which are disrupted at long range and in concert wit h disruption of the well characterized cytoplasmic interactions.