CRAC channel-based optogenetics

Abstract

Store-operated Ca²⁺ entry (SOCE) constitutes a major Ca²⁺ influx pathway in mammals to regulate a myriad of physiological processes, including muscle contraction, synaptic transmission, gene expression, and metabolism. In non-excitable cells, the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, composed of ORAI and stromal interaction molecules (STIM), constitutes a prototypical example of SOCE to mediate Ca²⁺ entry at specialized membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and the plasma membrane (PM). The key steps of SOCE activation include the oligomerization of the luminal domain of the ER-resident Ca²⁺ sensor STIM1 upon Ca²⁺ store depletion, subsequent signal propagation toward the cytoplasmic domain to trigger a conformational switch and overcome the intramolecular autoinhibition, and ultimate exposure of the minimal ORAI-activating domain to directly engage and gate ORAI channels in the plasma membrane. This exquisitely coordinated cellular event is also facilitated by the C-terminal polybasic domain of STIM1, which physically associates with negatively charged phosphoinositides embedded in the inner leaflet of the PM to enable efficient translocation of STIM1 into ER-PM MCSs. Here, we present recent progress in recapitulating STIM1-mediated SOCE activation by engineering CRAC channels with optogenetic approaches. These STIM1-based optogenetic tools make it possible to not only mechanistically recapture the key molecular steps of SOCE activation, but also remotely and reversibly control Ca²⁺-dependent cellular processes, inter-organellar tethering at MCSs, and transcriptional reprogramming when combined with CRISPR/Cas9-based genome-editing tools.

Keywords: CRISPR/Cas9; Ion channel; Membrane contact sites; ORAI; Optogenetics; Stromal interaction molecule.