Native Store-operated Ca2+ Influx Requires the Channel Function of Orai1 and TRPC1
Native Store-operated Ca2+ Influx Requires the Channel Function of Orai1 and TRPC1
复制标题
DOI：
10.1074/jbc.m808097200
复制
发表时间：
2009-04-10
影响因子：
4.8
通讯作者：
Muallem, Shmuel
中科院分区：
文献类型：
--
作者：
Kim, Min Seuk;Zeng, Weizhong;Muallem, Shmuel
随着STIM 1和Orai 1的发现以及STIM 1对TRPC和Orai 1通道的门控，一个中心问题是每个通道在天然钙库操纵的Ca 2+内流（SOC）中的作用。在这里，我们使用的策略敲低Orai 1和TRPC 1单独和组合和救援的小干扰RNA保护的突变体（sm）的smOrai 1和smTRPC 1证明，在人胚肾（HEK）细胞，救援SOC需要低水平的smOrai 1和smTRPC 1的共转染。孔突变体Orai 1（E106 Q）在存在或不存在TRPC 1的情况下不能拯救SOC，并且令人惊讶的是，孔突变体TRPC 1（F562 A）在存在或不存在Orai 1的情况下不能拯救SOC。TRPC 1通过TRPC 1（D 639 D，D 640 D）与STIM 1（K684 K，K685 K）之间的静电相互作用进行门控。引人注目的是，仅能被STIM 1（K684 E，K685 E）突变体拯救的通道死亡TRPC 1（D 639 K，D 640 K）仅在与Orai 1和STIM 1（K684 E，K685 E）一起表达时才能恢复SOC。因此，我们发现Orai 1和TRPC 1与HEK细胞中的天然STIM 1相互作用的相互要求。相反，Jurkat细胞中的SOC和CRAC电流被Orai 1的敲低抑制，但不受TRPC 1或TRPC 3的敲低影响。这些发现定义了HEK细胞中天然SOC的分子组成以及STIM 1-Orai 1-TRPC 1复合物在SOC活性中的作用。
With the discovery of STIM1 and Orai1 and gating of both TRPC and Orai1 channels by STIM1, a central question is the role of each of the channels in the native store-operated Ca2+ influx (SOCs). Here, we used a strategy of knockdown of Orai1 and of TRPC1 alone and in combination and rescue by small interfering RNA-protected mutants (sm) of smOrai1 and smTRPC1 to demonstrate that in human embryonic kidney (HEK) cells, rescue of SOCs required co-transfection of low levels of both smOrai1 and smTRPC1. The pore mutant Orai1(E106Q) failed to rescue the SOCs in the presence or absence of TRPC1 and, surprisingly, the pore mutant TRPC1(F562A) failed to rescue the SOCs in the presence or absence of Orai1. TRPC1 is gated by electrostatic interaction between TRPC1(D639D, D640D) with STIM1(K684K, K685K). Strikingly, the channel-dead TRPC1(D639K, D640K) that can be rescued only by the STIM1(K684E, K685E) mutant could restore SOCs only when expressed with Orai1 and STIM1( K684E, K685E). Accordingly, we found a mutual requirement of Orai1 and TRPC1 for their interaction with the native STIM1 in HEK cells. By contrast, SOC and the CRAC current in Jurkat cells were inhibited by knockdown of Orai1 but were not influenced by knockdown on TRPC1 or TRPC3. These findings define the molecular makeup of the native SOCs in HEK cells and the role of a STIM1-Orai1-TRPC1 complex in SOC activity.