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.