Institute of Biophysics, University of Linz, 4040 Linz, Austria.
Cell Calcium. 2013 Feb;53(2):139-51. doi: 10.1016/j.ceca.2012.11.005. Epub 2012 Dec 5.
As the molecular composition of calcium-release activated calcium (CRAC) channels has been unknown for two decades, elucidation of selective inhibitors has been considerably hampered. By the identification of the two key components of CRAC channels, STIM1 and Orai1 have emerged as promising targets for CRAC blockers. The aim of this study was to thoroughly characterize the effects of two selective CRAC channel blockers on currents derived from STIM1/Orai heterologoulsy expressed in HEK293 cells. The novel compounds GSK-7975A and GSK-5503A were tested for effects on STIM1 mediated Orai1 or Orai3 currents by whole-cell patch-clamp recordings and for the effects on STIM1 oligomerisation or STIM1/Orai coupling by FRET microscopy. To investigate their site of action, inhibitory effects of these molecules were explored using Orai pore mutants. The GSK blockers inhibited Orai1 and Orai3 currents with an IC(50) of approximately 4μM and exhibited a substantially slower rate of onset than the typical pore blocker La(3+), together with almost no current recovery upon wash-out over 4min. For the less Ca(2+)-selective Orai1 E106D pore mutant, I(CRAC) inhibition was significantly reduced. FRET experiments indicated that neither STIM1-STIM1 oligomerization nor STIM1-Orai1 coupling was affected by these compounds. These CRAC channel blockers are acting downstream of STIM1 oligomerization and STIM1/Orai1 interaction, potentially via an allosteric effect on the selectivity filter of Orai. The elucidation of these CRAC current blockers represents a significant step toward the identification of CRAC channel-selective drug compounds.
由于钙释放激活钙 (CRAC) 通道的分子组成在过去二十年中一直未知，因此选择性抑制剂的阐明受到了相当大的阻碍。通过鉴定 CRAC 通道的两个关键组成部分，STIM1 和 Orai1 已成为 CRAC 阻滞剂的有前途的靶标。本研究的目的是彻底表征两种选择性 CRAC 通道阻滞剂对异源表达于 HEK293 细胞中的 STIM1/Orai 衍生电流的影响。通过全细胞膜片钳记录测试了新型化合物 GSK-7975A 和 GSK-5503A 对 STIM1 介导的 Orai1 或 Orai3 电流的影响，以及对 FRET 显微镜下的 STIM1 寡聚化或 STIM1/Orai 偶联的影响。为了研究它们的作用部位，通过 Orai 孔突变体探索了这些分子的抑制作用。这些 GSK 阻滞剂以约 4μM 的 IC50 抑制 Orai1 和 Orai3 电流，与典型的孔阻滞剂 La(3+) 相比，其起始速度要慢得多，并且在 4 分钟以上的冲洗过程中几乎没有电流恢复。对于 Ca2+ 选择性较低的 Orai1 E106D 孔突变体，I(CRAC)抑制作用显著降低。FRET 实验表明，这些化合物既不影响 STIM1-STIM1 寡聚化，也不影响 STIM1-Orai1 偶联。这些 CRAC 通道阻滞剂作用于 STIM1 寡聚化和 STIM1/Orai1 相互作用的下游，可能通过对 Orai 选择性过滤器的变构作用。这些 CRAC 电流阻滞剂的阐明代表了朝着鉴定 CRAC 通道选择性药物化合物迈出的重要一步。
