Wootton Laura L, Michelangeli Francesco
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
J Biol Chem. 2006 Mar 17;281(11):6970-6. doi: 10.1074/jbc.M510978200. Epub 2006 Jan 6.
Three isoforms of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) are known to exist in mammalian cells. This study investigated the effects of thapsigargin and a variety of commonly used hydrophobic inhibitors on these SERCA isoforms (i.e. SERCA1b, SERCA2b, and SERCA3a), which were transiently expressed in COS-7 cells. In addition, the study assessed whether the introduction of the phenylalanine to valine mutation at position 256 (F256V), known to reduce the potency of thapsigargin inhibition in avian SERCA1, affects the other SERCA isoforms in a similar manner and whether this mutation also affects the inhibition by other inhibitors. This study has shown that the sensitivity to thapsigargin is different for the SERCA isoforms (apparent K(i) values being 0.21, 1.3, and 12 nm for SERCA1b, SERCA2b, and SERCA3a, respectively). The reduction in thapsigargin sensitivity caused by the F256V mutation was also different for the three isoforms, with SERCA2b only being modestly affected by this mutation. Although some of the other inhibitors investigated (i.e. cyclopiazonic acid and curcumin) showed some differences in their sensitivity toward the SERCA isoforms, most were little affected by the F256V mutation, indicating that they inhibit the Ca(2+)-ATPase by binding to sites on SERCA distinct from that of thapsigargin.
已知在哺乳动物细胞中存在三种肌浆网/内质网Ca(2+)ATP酶（SERCA）亚型。本研究调查了毒胡萝卜素和多种常用疏水性抑制剂对这些在COS-7细胞中瞬时表达的SERCA亚型（即SERCA1b、SERCA2b和SERCA3a）的影响。此外，该研究评估了在256位引入苯丙氨酸到缬氨酸的突变（F256V）——已知该突变会降低毒胡萝卜素对禽类SERCA1的抑制效力——是否以类似方式影响其他SERCA亚型，以及该突变是否也会影响其他抑制剂的抑制作用。本研究表明，SERCA亚型对毒胡萝卜素的敏感性不同（SERCA1b、SERCA2b和SERCA3a的表观K(i)值分别为0.21、1.3和12 nM）。F256V突变引起的毒胡萝卜素敏感性降低在三种亚型中也有所不同，其中SERCA2b仅受到该突变的适度影响。尽管所研究的一些其他抑制剂（即环匹阿尼酸和姜黄素）对SERCA亚型的敏感性存在一些差异，但大多数受F256V突变的影响较小，这表明它们通过与SERCA上与毒胡萝卜素不同的位点结合来抑制Ca(2+)-ATP酶。
