골격근 근장그물 칼슘이동에 대한 Phospholamban 펩타이드의 조절

Effect of a Phospholamban Peptide on the Skeletal Sarcoplasmic Reticulum Ca²⁺ Transport

Phospholamban은 심근 근장그물 Ca²⁺-ATPase 조절단백이다. 조절작용기전은 탈인산화된 phospholamban에 의해 Ca²⁺-ATPase가 억제됨으로 나타나며, 이 phospholamban이 인산화됨으로 Ca²⁺-ATPase에 대한 억제가 반전됨을 보인다. 최근에 phospholamban의 cytoplasmic domain만으로 Ca²⁺-ATPase를 억제하기에는 불충분하다는 보고가 있어 본 실험을 계획하였다. Ca²⁺-ATPase의 활성을 억제하는 phospholamban domain을 밝히기 위하여 합성한 phospholamban 펩타이드(아미노산 1-25)의 Ca²⁺ uptake에 대한 효과를 살펴보았다. 골격근 근장그물에서 Ca²⁺-ATPase를 분리한 후 phosphatidylcholine이나 phosphatidylcholine과 phosphatidylserine을 포함한 liposome에 재조합시켰다. Phospholamban 펩타이드는 phosphatidylcholine을 이용하여 재조합된 vesicles의 초기 Ca²⁺ uptake rate를 억제하고, cAMP 의존성 protein kinase의 catalytic subunit로 인산화시킨 phospholamban 펩타이드는 이 억제를 반전시킴을 보여 주었다. Phosphatidylcholine과 phosphatidylserine을 포함한 제조합 vesicles에서도 같은 양상을 보였다. 이상의 결과로 미루어 볼 때 인산화 sites를 포함하고 있는 phospholamban의 cytoplasmic domain은 그 자체만으로도 근장그물 칼슘펌프를 억제하기에 충분하다고 결론지을 수 있다.

Phospholamban is the regulator of Ca²⁺-ATPase in cardiac sarcoplasmic reticulum(SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban. Phosphorylation of phospholamban relieves this inhibition. Recently, there has been a report that the cytoplasmic domain (amino acids 1-25) of phospholamban is insufficient to inhibit the Ca²⁺ pump. To explore the domains of phospholamban responsible for Ca²⁺-ATPase inhibitory activity, we examined the effect of a synthetic phospholamban peptide consisting of amino acid residues 1-25 on Ca²⁺ uptake by reconstituted skeletal SR Ca²⁺-ATPase. The Ca²⁺-ATPase of skeletal SR was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC) or phosphatidylcholine: phosphatidylserine (PC:PS). Inclusion of a phospholamban peptide in PC proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺ uptake at pCa 6.0, and phosphorylation of this peptide by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effect on the Ca²⁺ pump. Similar effects of phospholamban peptide were also observed using PC:PS proteoliposomes. Based on these results, we could conclude that the cytoplasmic domain of phospholamban, containing the phosphorylation sites, by itself is sufficient to inhibit the Ca²⁺ pump of SR.