Regulation of the Skeletal Sarcoplasmic Reticulum Ca²⁺ Pump by Phospholamban in Reconstituted Phospholipid Vesicles

Abstract

Phospholamban is the regulator of the Ca²⁺-ATPase in cardiac sarcoplasmic reticulum (SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban, and phosphorylation may relieve this inhibition. Fast-twitch skeletal muscle SR does not contain phospholamban, and it is not known whether the Ca²⁺-ATPase isoform from this muscle may be also subject to regulation by phospholamban in a similar manner as the cardiac isoform. To determine this we reconstituted the skeletal isoform of the SR Ca²⁺-ATPase with phospholamban in phosphatidylcholine proteoliposomes. Inclusion of phospholamban was associated with significant inhibition of the initial rates of Ca²⁺ uptake at pCa 6.0, and phosphorylation of phospholamban by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects on the Ca²⁺ pump. Similar effects of phospholamban were also observed using phosphatidylcholine: phosphatidylserine proteoliposomes, in which the Ca²⁺ pump was activated by the negatively charged phospholipids (24). Regulation of the Ca²⁺-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by cross-linking experiments, using a synthetic peptide that corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca²⁺-ATPase may be also regulated by phospholamban, although this regulator is not expressed in fast-twitch skeletal muscles.

Key Words:

• Muscle
• sarcoplasmic reticulum
• Ca²⁺-pump
• phospholamban
• reconstitution
• cross-linker
• photoaffinity labeling