A comparative functional analysis of plasma membrane Ca2+pump isoforms in intact cells
A comparative functional analysis of plasma membrane Ca2+pump isoforms in intact cells
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DOI：
10.1074/jbc.m300784200
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发表时间：
2003-07-04
影响因子：
4.8
通讯作者：
Carafoli, E
中科院分区：
文献类型：
--
作者：
Brini, M;Coletto, L;Carafoli, E
质膜 Ca2+ 泵的四种基本亚型和两个 C 端截短的剪接变体 PMCA4CII(4a) 和 3CII(3a) 在中国仓鼠卵巢细胞中与靶向细胞质、内质网和线粒体的水母发光蛋白一起瞬时过表达。由于 PMCA3CII(3a) 尚未被克隆和研究，因此本研究对其进行了克隆、部分纯化和表征。与先前研究的 PMCA4 的相应截短变体不同，PMCA3CII(3a) 具有非常高的钙调蛋白亲和力。所有四种基本泵变体都会影响天然细胞内环境中 Ca2+ 的稳态。内质网中的[Ca2+]水平以及由于肌醇1,4,5-三磷酸清空内质网储存而在细胞质和线粒体中产生的[Ca2+]瞬变高度均因泵的过度表达而降低。神经元特异性 PMCA2 和 PMCA3 的效果比普遍表达的亚型 1 和 4 的效果要大得多。出乎意料的是，截短的 PMCA3 和 PMCA4 在影响细胞质和细胞器中 Ca2+ 稳态方面与全长变体一样有效。特别是，PMCA4CII(4a) 与 PMCA4CI(4b) 一样有效，即使其对钙调蛋白的亲和力低得多。结果表明，钙调蛋白的可用性对于体内 PMCA 泵的调节可能并不重要。
The four basic isoforms of the plasma membrane Ca2+ pump and the two C-terminally truncated spliced variants PMCA4CII(4a) and 3CII(3a) were transiently over-expressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum, and the mitochondria. As PMCA3CII(3a) had not yet been cloned and studied, it was cloned for this study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously, PMCA3CII( 3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca2+ in the native intracellular environment. The level of [Ca2+] in the endoplasmic reticulum and the height of the [Ca2+] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full-length variants in influencing the homeostasis of Ca2+ in the cytosol and the organelles. In particular, PMCA4CII( 4a) was as effective as PMCA4CI(4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo.