Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca(2+)-ATPase in 
cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the 
enzyme for Ca(2+). Recent cross-linking studies have suggested that PLB binding 
and Ca(2+) binding to SERCA2a are mutually exclusive. PLB binds to the E2 
conformation of the Ca(2+)-ATPase, preventing formation of E1, the conformation 
that binds two Ca(2+) (at sites I and II) with high affinity and is required for 
ATP hydrolysis. Here we determined whether Ca(2+) binding to site I, site II, or 
both sites is sufficient to dissociate PLB from the Ca(2+) pump. Seven SERCA2a 
mutants with amino acid substitutions at Ca(2+)-binding site I (E770Q, T798A, 
and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) 
were made, and the effects of Ca(2+) on N30C-PLB cross-linking to Lys(328) of 
SERCA2a were measured. In agreement with earlier reports with the skeletal 
muscle Ca(2+)-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP 
in the presence of Ca(2+); however, all were phosphorylatable by P(i) to form 
E2P. Ca(2+) inhibition of E2P formation was observed only in SERCA2a mutants 
retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB 
and each Ca(2+)-ATPase mutant was observed in the absence of Ca(2+). 
Importantly, however, micromolar Ca(2+) inhibited PLB cross-linking only to 
mutants retaining a functional Ca(2+)-binding site I. The dynamic equilibrium 
between Ca(2+) pumps and N30C-PLB was retained by all mutants, demonstrating 
normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. 
From these results we conclude that site I is the key Ca(2+)-binding site 
regulating the physical association between PLB and SERCA2a.