Crosslinking unnatural amino acids unravel peripheral channel dynamics critical for Orai1 pore opening

Description

As Calcium (Ca2+) plays a major role in a variety of cellular processes such as gene transcription, proliferation, or apoptosis, the dysregulation of Ca2+ homeostasis can result in a variety of pathological conditions like developmental issues, cancer, and immune deficiencies. On of the most crucial Ca2+ entry pathways is the ubiquitously expressed Ca2+ release-activated Ca2+ (CRAC) channel. This specific ion channel is 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 demonstrated 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 as well as its exact binding sites of STIM1 still remain elusive. Therefore, via a combined approach of electrophysiology, Ca2+ imaging, confocal microscopy 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 want to resolve the critical positions and dynamics involved in the CRAC-channel activation. Here, our focus is on the channel periphery as well as in the cytosolic helical extension of Orai1. Especially the application of the photocrosslinking UAAs azido-phenylalanine (Azi) and benzo-phenylalanine (Bpa) as well as chemical- crosslinking UAAs helped us to gain a better resolution of the structure/function relationship of the ion channel on an amino acid level and we were able to identify several positions critical for STIM1 mediated pore opening. Our current investigation deals with the nexus region (L261-K265), as it is an important site for channel gating, STIM1 coupling and pore opening and its mutation to ANSGA results in constitutive activity. Via the incorporation of different UAAs we want to gain a better understanding of the exact role of this particular region in the opening mechanism of the CRAC channel machinery.
(Austrian Science Fund (FWF) projects 32851, 35900 and 36202 to I.D.; 6871323 to A.T.)

Period	18 Feb 2025
Event title	Biophysical Society Meeting 2025
Event type	Conference
Location	Los Angeles, United StatesShow on map
Degree of Recognition	International