Asanov Alexander, Sampieri Alicia, Moreno Claudia, Pacheco Jonathan, Salgado Alfonso, Sherry Ryan, Vaca Luis
TIRF Labs, 106 Grendon Place, Cary, NC 27519, USA.
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, México, DF 04510, Mexico.
Cell Calcium. 2015 Jan;57(1):1-13. doi: 10.1016/j.ceca.2014.10.011. Epub 2014 Oct 31.
Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.
细胞内钙离子储存的耗尽引发了一系列快速的事件，最终导致质膜上所谓的储存-操纵通道（SOC）的激活。通过SOC的钙内流对于启动钙依赖性细胞内信号传导以及重新填充内部钙储存至关重要，从而确保信号级联的再生。尽管这种进化保守机制具有重要意义，但SOC的分子身份在过去20年一直是激烈争论的焦点。最初的研究将瞬时受体电位经典（TRPC）通道超家族的一些成员（更有力的证据指向TRPC1）定位为假定的SOC。最近的证据表明，基质相互作用分子1（STIM1）激活了TRPC通道家族的一些成员。然而，TRPC通道的确切亚基组成至今仍未确定。为了确定STIM1激活的TRPC通道的亚基组成，我们开发了一种新方法，该方法将基于膜片钳技术的单通道电生理测量与单分子荧光成像相结合。我们将这种方法称为单离子通道单分子检测技术（SC-SMD）。使用SC-SMD方法，我们获得了STIM1激活的TRPC通道亚基组成的直接证据。此外，我们的电生理成像SC-SMD方法在分子水平上提供了STIM1和钙调蛋白拮抗调节TRPC通道活性机制的证据。
