Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs)

J Biol Chem. 2002 Jul 26;277(30):26725-8. doi: 10.1074/jbc.C200269200. Epub 2002 May 24.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Calcium / metabolism
  • Calcium-Binding Proteins / metabolism*
  • Calcium-Transporting ATPases / antagonists & inhibitors*
  • Cell Line
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Inhibitors / pharmacology*
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes
  • Humans
  • Kinetics
  • Microsomes / metabolism
  • Muscle Fibers, Fast-Twitch / enzymology
  • Muscle Proteins / physiology*
  • Muscle, Skeletal / enzymology
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Proteolipids / physiology*
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases
  • Time Factors
  • Transfection

Substances

  • Calcium-Binding Proteins
  • DNA, Complementary
  • Enzyme Inhibitors
  • Epitopes
  • Muscle Proteins
  • Proteolipids
  • phospholamban
  • sarcolipin
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases
  • Calcium-Transporting ATPases
  • Calcium