Characterization of the role of inner TM segments and cytosolic helical TM extension in STIM1-mediated Orai1 pore opening

Description

In the human body Calcium (Ca2+) plays a major role in a variety of cellular processes such as gene transcription, proliferation, or apoptosis. Hence, a dysregulation of the Ca2+ homeostasis might result in severe conditions such as developmental issues, cancer, and immune deficiencies. Here, the ubiquitously expressed Ca2+ release-activated Ca2+ (CRAC) channel has been identified as one of the most crucial Ca2+ entry pathways. This specific ion channel is fully composed of two proteins, the Ca2+ sensor located in the endoplasmic reticulum (ER), Stromal Interaction Molecule (STIM1), and the ion conducting pore Orai1 situated in the plasma membrane (PM). Upon depletion of the intracellular Ca2+ stores STIM1 activates, oligomerizes, translocates to ER-PM junctions and activates Orai1. The coupling of STIM1 to Orai1 and the consequent signal propagation from the channel periphery all the way to the pore finally results in Ca2+ influx. Our previous findings stress the significance of specific Orai1 gating checkpoints inducing a local conformational change resulting in an overall global

transmembrane (TM) domain movement essential for pore opening. However, the precise underlying dynamics within the individual TM domains of Orai1 still remain elusive. Therefore, via a combined approach of Ca2+ imaging, electrophysiology, and molecular dynamic (MD) simulations together with conventional site directed mutagenesis and the incorporation of unnatural amino acids (UAAs) via genetic code expansion (GCE), we offer an overview of critical sites and dynamics in the channel periphery as well as in the cytosolic helical extension essential for pore opening. Especially the application of the photocrosslinking UAAs azido-phenylalanine (Azi) and benzo-phenylalanine (Bpa) helped us to gain a better resolution of the structure/function relationship of the ion channel on an amino acid level. Here, we identified and characterized the position V181 in TM3 as critical site in STIM1 mediated pore opening.
(Austrian Science Fund (FWF) projects 32851, 35900 and 36202 to I.D.; 6871323 to A.T.)

Period	30 Jun 2024 → 05 Jul 2024
Event title	Gordon Research Conference: Calcium Signaling
Event type	Conference
Location	Lucca, ItalyShow on map
Degree of Recognition	International