Mignen Olivier, Thompson Jill L, Shuttleworth Trevor J
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA.
J Physiol. 2009 Sep 1;587(Pt 17):4181-97. doi: 10.1113/jphysiol.2009.174193. Epub 2009 Jul 21.
The activation of Ca(2+) entry is a critical component of agonist-induced cytosolic Ca(2+) signals in non-excitable cells. Although a variety of different channels may be involved in such entry, the recent identification of the STIM and Orai proteins has focused attention on the channels in which these proteins play a key role. To date, two distinct highly Ca(2+)-selective STIM1-regulated and Orai-based channels have been identified - the store-operated CRAC channels and the store-independent arachidonic acid activated ARC channels. In contrast to the CRAC channels, where the channel pore is composed of only Orai1 subunits, both Orai1 and Orai3 subunits are essential components of the ARC channel pore. Using an approach involving the co-expression of a dominant-negative Orai1 monomer along with different preassembled concatenated Orai1 constructs, we recently demonstrated that the functional CRAC channel pore is formed by a homotetrameric assembly of Orai1 subunits. Here, we use a similar approach to demonstrate that the functional ARC channel pore is a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits. Expression of concatenated pentameric constructs with this stoichiometry results in the appearance of large currents that display all the key biophysical and pharmacological features of the endogenous ARC channels. They also replicate the essential regulatory characteristics of native ARC channels including specific activation by low concentrations of arachidonic acid, complete independence of store depletion, and an absolute requirement for the pool of STIM1 that constitutively resides in the plasma membrane.
钙离子内流的激活是激动剂诱导的非兴奋性细胞胞质钙离子信号的关键组成部分。尽管多种不同的通道可能参与这种内流，但近期对基质相互作用分子（STIM）和奥拉帕蛋白（Orai）的鉴定使人们将注意力集中在这些蛋白发挥关键作用的通道上。迄今为止，已鉴定出两种截然不同的、高度钙离子选择性的、由STIM1调节且基于Orai的通道——储存操纵性钙释放激活钙通道（CRAC通道）和不依赖储存的花生四烯酸激活的ARC通道。与CRAC通道不同，CRAC通道的孔仅由Orai1亚基组成，而Orai1和Orai3亚基都是ARC通道孔的必需组成部分。我们最近采用一种方法，将显性负性Orai1单体与不同的预组装串联Orai1构建体共表达，结果表明功能性CRAC通道孔是由Orai1亚基的同四聚体组装形成的。在此，我们采用类似方法证明功能性ARC通道孔是由三个Orai1亚基和两个Orai3亚基组成的异五聚体组装。以这种化学计量比表达串联五聚体构建体，会出现大电流，这些电流表现出内源性ARC通道的所有关键生物物理和药理学特征。它们还重现了天然ARC通道的基本调节特性，包括低浓度花生四烯酸的特异性激活、完全不依赖储存耗竭以及对组成性存在于质膜中的STIM1池的绝对需求。
