Amino acids Glu2 to Ile18 in the cytoplasmic domain of phospholamban are essential for functional association with the Ca(2+)-ATPase of sarcoplasmic reticulum.

Abstract

Phospholamban inhibits the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum by lowering its affinity for Ca2+. In earlier studies (Toyofuku, T., Kazimierz, K., Tada, M., and MacLennan, D. H. (1993) J. Biol. Chem. 268, 2809-2815), parts of the phosphorylation and nucleotide binding/hinge domains of the Ca(2+)-ATPase were shown to be essential for phospholamban interaction. In order to identify the sites in phospholamban which interact with the Ca(2+)-ATPase, a series of mutants containing amino acid replacements in the cytoplasmic and transmembrane regions of phospholamban were co-expressed with the cardiac/slow-twitch muscle Ca(2+)-ATPase isozyme, SERCA2a, in HEK-293 cells. Mutation of residues in the cytoplasmic 1A domain of phospholamban, including positively charged residues, Lys3, Arg9, Arg13, and Arg14, negatively charged residue, Glu2, hydrophobic residues, Val4, Leu7, Ala11, Ile12, Ala15, and Ile18, and phosphorylation site residues, Ser16 and Thr17, resulted in loss of the inhibitory effect of phospholamban on Ca2+ transport by the Ca(2+)-ATPase. By contrast, mutation of Met1, Gln5, Tyr6, Thr8, Ser10, Glu19, or Met20 or of residues in the cytoplasmic 1B domain (Pro21 to Asn30) and of Cys41 in the transmembrane domain (Leu31 to Leu52) did not diminish the inhibitory effects of phospholamban on Ca2+ transport. These results suggest that a region essential for functional association of phospholamban with the Ca(2+)-ATPase lies in the cytoplasmic 1A domain of phospholamban.