Ca 2+ ATPase Phospholamban, Sarcolipin Publications Brody Disease Cardiomyopathy Ca2+ Release Channel MH/CCD Ca2+ Binding Proteins OMIM SERCA2 OMIM PLN OMIM SLN						Interactions of SERCA Molecules with Phospholamban and Sarcolipin SERCA2a, the cardiac/slow-twitch isoform binds to and is regulated by phospholamban (PLN), a 52 amino acid, homopentameric protein made up of three domains: cytoplasmic domain Ia is a helical, charged sequence of 20 amino acids, containing sites for phosphorylation by protein kinase A and calmodulin kinase; domain Ib is an unstructured, polar, 10 amino acid sequence; and domain II is a transmembrane helix made up of of 22 hydrophobic amino acids. In its dephospho form, PLN inhibits SERCA2a activity by physical interactions which lower the affinity of SERCA2a for Ca2+: when phosphorylated, the SERCA2a binding and inhibitory properties of PLN are lost. We are carrying out a program to define the sites of interaction between SERCA2a and PLN, to determine the mechanism of inhibition and to define the role of PLN monomers and pentamers in inhibitory interactions. We use many of the tools used for structure/function analysis of SERCA1 molecules, but, in this case, we coexpress the two molecules and measure Ca2+ dependence of Ca2+ transport as a measure of inhibitory interactions. We have defined the sites of noninhibitory interaction in the cytoplasmic sequences of the two molecules. These involve short sequences of charged and hydrophobic residues in each molecule. By expressing SERCA2a with only the transmembrane sequence of PLN, we demonstrated that inhibitory interactions occur in the transmembrane domains. As in SERCA molecules alone, long range interactions occur in the SERCA2a/PLN complex. When the non inhibitory, cytoplasmic interactions are disrupted by phosphorylation, the transmembrane inhibitory interactions are also disrupted: when the transmembrane inhibitory interactions are disrupted by Ca2+binding, the non inhibitory, cytoplasmic interactions are also disrupted. We have used alanine scanning mutagenesis to demonstrate that the PLN transmembrane helix contains one face involved in PLN/PLN interaction to form pentamers and another face involved in PLN/SERCA2a heterodimer formation. Since mutations that increase monomer formation 3 to 4 fold also increase inhibitory function 3 fold, we have deduced that the PLN monomer is the inhibitory species. Our current work is aimed at refining these concepts and at determining the PLN interaction sites in the transmembrane domain of SERCA2a. We discovered that a proteolipid, now referred to as sarcolipin (SLN), is associated with SERCA1a.  We cloned DNA encoding this protein, and, through coexpression, we have shown that sarcolipin, like phospholamban, lowers the affinity of SERCA1 for Ca2+, but at saturating calcium concentrations, increases Vmax by 40%.
Ca 2+ ATPase Phospholamban, Sarcolipin Top of Page Brody Disease Cardiomyopathy Ca2+ Release Channel MH/CCD Ca2+ Binding Proteins OMIM SERCA2 OMIM PLN OMIM SLN						Phospholamban/Sarcolipin Publications MacLennan, D.H., Kimura, Y. and Toyofuku, T. (1998) Sites of regulatory interaction between calcium ATPases and phospholamban. Annals of the New York Academy of Science (in press) Odermatt, A., Becker, S., Khanna, V.K., Kurzydlowski, K., Leisner, E., Pette, D. and MacLennan, D.H. (1998) Sarcolipin regulates the activity of SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase. Journal of Biological Chemistry 273(in press) Odermatt, A., Taschner, P.E., Scherer, S.W., Beatty, B., Khanna, V.K., Cornblath, D.R., Chaudhry, V., Yee, W.C., Schrank, B., Karpati, G., Breuning, M.H., Knoers, N. and MacLennan, D.H. (1997) Characterization of the gene encoding human sarcolipin (SLN), a proteolipid associated with SERCA1: absence of structural mutations in five patients with Brody disease. Genomics 45(3):541-53 Kimura, Y., Kurzydlowski, K., Tada, M. and MacLennan, D.H. (1997) Phospholamban inhibitory function is activated by depolymerization. Journal of Biological Chemistry 272(24):15061-4 Kimura, Y., Kurzydlowski, K., Tada, M. and MacLennan, D.H. (1996) Phospholamban regulates the Ca2+-ATPase through intramembrane interactions. Journal of Biological Chemistry 271(36):21726-31 Toyofuku, T., Kurzydlowski, K., Tada, M. and MacLennan, D.H. (1994) Amino acids Lys-Asp-Asp-Lys-Pro-Val402 in the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum are critical for functional association with phospholamban. Journal of Biological Chemistry 269(37):22929-32 Toyofuku, T., Kurzydlowski, K., Tada, M. and MacLennan, D.H. (1994) Amino acids Glu2 to Ile18 in the cytoplasmic domain of phospholamban are essential for functional association with the Ca(2+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 269(4):3088-94 Toyofuku, T., Kurzydlowski, K., Tada, M. and MacLennan, D.H. (1993) Identification of regions in the Ca(2+)-ATPase of sarcoplasmic reticulum that affect functional association with phospholamban. Journal of Biological Chemistry 268(4):2809-15 MacLennan, D.H. and Toyofuku, T. (1992) Structure-function relationships in the Ca2+ pump of the sarcoplasmic reticulum. Biochemical Society Transactions 20(3):559-62 Fujii, J., Zarain-Herzberg, A., Willard, H.F., Tada, M. and MacLennan, D.H. (1991) Structure of the rabbit phospholamban gene, cloning of the human cDNA, and assignment of the gene to human chromosome 6. Journal of Biological Chemistry 266(18):11669-75 Fujii, J., Maruyama, K., Tada, M. and MacLennan, D.H. (1990) Co-expression of slow-twitch/cardiac muscle Ca2(+)-ATPase (SERCA2) and phospholamban. FEBS Letters 273(1-2):232-4 Fujii, J., Maruyama, K., Tada, M. and MacLennan, D.H. (1989) Expression and site-specific mutagenesis of phospholamban. Studies of residues involved in phosphorylation and pentamer formation. Journal of Biological Chemistry 264(22):12950-5 Fujii, J., Lytton, J., Tada, M. and MacLennan, D.H. (1988) Rabbit cardiac and slow-twitch muscle express the same phospholamban gene. FEBS Letters 227(1):51-5
Phospholamban, Sarcolipin Top of Page Publications Revised 1998Apr09