Haynes Lee P, Tepikin Alexei V, Burgoyne Robert D
The Physiological Laboratory, Crown Street, University of Liverpool, Liverpool L69 3BX, UK.
J Biol Chem. 2004 Jan 2;279(1):547-55. doi: 10.1074/jbc.M309617200. Epub 2003 Oct 21.
Intracellular calcium signals are responsible for initiating a spectrum of physiological responses. The caldendrins/calcium-binding proteins (CaBPs) represent mammal-specific members of the CaM superfamily. CaBPs display a restricted pattern of expression in neuronal/retinal tissues, suggesting a specialized role in Ca2+ signaling in these cell types. Recently, it was reported that a splice variant of CaBP1 functionally interacts with inositol 1,4,5-trisphosphate (InsP3) receptors to elicit channel activation in the absence of InsP3 (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711-7716). These data indicate a new mode of InsP3 receptor modulation and hence control of intracellular Ca2+ concentration ([Ca2+]i) in neuronal tissues. We have analyzed the biochemistry of the long form splice variant of CaBP1 (L-CaBP1) and show that, in vitro, a recombinant form of the protein is able to bind Ca2+ with high affinity and undergo a conformational change. We also describe the localization of endogenous and overexpressed L-CaBP1 in the model neuroendocrine PC12 cell system, where it was associated with the plasma membrane and Golgi complex in a myristoylation-dependent manner. Furthermore, we show that overexpressed L-CaBP1 is able to substantially suppress rises in [Ca2+]i in response to physiological agonists acting on purinergic receptors and that this inhibition is due in large part to blockade of release from intracellular Ca2+ stores. The related protein neuronal calcium sensor-1 was without effect on the [Ca2+]i responses to agonist stimulation. Measurement of [Ca2+] within the ER of permeabilized PC12 cells demonstrated that LCaBP1 directly inhibited InsP3-mediated Ca2+ release. Expression of L-CaBP1 also inhibited histamine-induced [Ca2+]i oscillations in HeLa cells. Together, these data suggest that L-CaBP1 is able to specifically regulate InsP3 receptor-mediated alterations in [Ca2+]i during agonist stimulation.
细胞内钙信号负责引发一系列生理反应。钙结合蛋白（CaBPs）是钙调蛋白超家族中哺乳动物特有的成员。CaBPs在神经元/视网膜组织中呈现出有限的表达模式，表明其在这些细胞类型的Ca2+信号传导中具有特殊作用。最近，有报道称CaBP1的一种剪接变体在没有肌醇1,4,5-三磷酸（InsP3）的情况下与InsP3受体发生功能相互作用，从而引发通道激活（Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711-7716）。这些数据表明了InsP3受体调节的一种新模式，进而控制神经元组织中的细胞内Ca2+浓度（[Ca2+]i）。我们分析了CaBP1长形式剪接变体（L-CaBP1）的生物化学性质，结果表明，在体外，该蛋白的重组形式能够以高亲和力结合Ca2+并发生构象变化。我们还描述了内源性和过表达的L-CaBP1在模型神经内分泌PC12细胞系统中的定位，在该系统中，它以肉豆蔻酰化依赖的方式与质膜和高尔基体复合体相关联。此外，我们表明过表达的L-CaBP1能够显著抑制因作用于嘌呤能受体的生理激动剂而导致的[Ca2+]i升高，并且这种抑制在很大程度上是由于对细胞内Ca2+储存释放的阻断。相关蛋白神经元钙传感器-1对激动剂刺激引起的[Ca2+]i反应没有影响。对通透PC12细胞内质网内Ca2+的测量表明，L-CaBP1直接抑制InsP3介导的Ca2+释放。L-CaBP1的表达也抑制了HeLa细胞中组胺诱导的[Ca2+]i振荡。总之，这些数据表明L-CaBP1能够在激动剂刺激期间特异性调节InsP3受体介导的[Ca2+]i变化。
