Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs).
Sarcolipin (SLN), a regulator of the sarco(endo)plasmic reticulum Ca(2+)-ATPase of fast-twitch skeletal muscle (SERCA1a), is also expressed in cardiac and slow-twitch skeletal muscles where phospholamban (PLN) and SERCA2a are expressed. Co-expression in HEK-293 cells of SLN tagged N-terminally with a FLAG epitope (NF-SLN), PLN, and SERCAs followed by measurement of the Ca(2+) dependence of Ca(2+) transport activity in isolated microsomal fractions showed that NF-SLN can reduce the apparent Ca(2+) affinity of both SERCA1a (DeltaK(Ca) = -0.22 +/- 0.01 pCa units) and SERCA2a (DeltaK(Ca) = -0.37 +/- 0.04 pCa units). When SERCA1a or SERCA2a were co-expressed with both NF-SLN and PLN, inhibition was synergistic, reducing DeltaK(Ca) by about -1.0 pCa units. Co-immunoprecipitation showed that NF-SLN increased the binding of PLN to SERCA, whereas PLN did not increase the binding of NF-SLN to SERCA. Elevated Ca(2+) dissociates both PLN and NF-SLN from their complexes with both SERCA1a and SERCA2a, but NF-SLN induced resistance to Ca(2+) dissociation of the PLN.SERCA complex. Co-immunoprecipitation of PLN and NF-SLN without SERCA showed that NF-SLN binds directly to PLN and that NF-SLN inhibits the formation of PLN pentamers. Thus the ability of NF-SLN to elevate the content of PLN monomers can account, at least in part, for the superinhibitory effects of NF-SLN in the presence of PLN.
Asahi
Michio
M
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Kurzydlowski
Kazimierz
K
Tada
Michihiko
M
MacLennan
David H
DH
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology; D009124:Muscle Proteins / Q000502:physiology*; D018482:Muscle, Skeletal / Q000201:enzymology; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000502:physiology*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013997:Time Factors; D014162:Transfection; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D000939:Epitopes; D009124:Muscle Proteins; D011510:Proteolipids; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C077258:sarcolipin