Defects in the STIM1 SOARα2 domain affect multiple steps in the CRAC channel activation cascade

The calcium release-activated calcium (CRAC) channel consists of STIM1, alpha -Ca2+ sensor in the endoplasmic reticulum (ER), and Orai1, the Ca2+ ion channel in the plasma membrane. Ca2+ store depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 alpha 2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1 alpha 1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners.

Automated summary

The CRAC channel is composed of STIM1 and Orai1, which interact with each other upon Ca2+ store depletion. Mutations at F394 in STIM1 alpha 2 have diverse effects on STIM1 activation steps, affecting homomerization and subsequent Orai1 activation. Simulation results suggest that these mutations impact the STIM1/Orai1 cascade through altered protein-protein interactions and potential electrostatic interactions with lipid headgroups in the ER membrane.