The Multifaceted Mechanisms of Orai Channel Gating
Open Access
- Author:
- Jennette, Michelle
- Graduate Program:
- Anatomy
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 08, 2023
- Committee Members:
- Patricia Mclaughlin, Major Field Member
Loren Evey, Major Field Member
Jeffrey Neighbors, Outside Unit & Field Member
Scot Kimball, Major Field Member
Christopher Yengo, Co-Chair & Dissertation Advisor
Charles Lang, Outside Field Member
Donald Gill, Co-Chair & Dissertation Advisor
Patricia Mclaughlin, Program Head/Chair - Keywords:
- Orai
STIM
Calcium
Calcium signalling
Gain of function - Abstract:
- Store-operated Ca2+ entry (SOCE) is a fundamental homeostatic mechanism that controls intracellular calcium (Ca2+) concentrations in non-excitable cells. Ca2+ must be tightly regulated, as it mediates numerous physiological processes including gene transcription, proliferation, muscle contraction, neurotransmitter release, and apoptosis. After ligand-induced store depletion, decreased Ca2+ concentrations in the ER lumen are detected by the single pass Ca2+ sensing protein, stromal interaction molecule (STIM). Specifically, Ca2+ dissociates from the N-terminal EF-hand domains of STIM, which causes them to pinch together and dimerize. This triggers a large conformational change in the cytoplasmic C-terminus of the STIM molecule, where the two single pass TMs and CC1 domains zip together and oligomerize, causing the STIM-Orai activating region (SOAR) to flip out. Further, while STIM migrates into ER-PM junctions, it also extends the full 15 nm gap to bind hexameric plasma membrane (PM)-located Orai channels. The binding of SOAR to ion selective Orai channels results in the flow of Ca2+ into the cell to replenish ER stores or to be used for different signaling pathways. Understanding the Orai-STIM coupling mechanism has been a focus for many biochemical studies, with multiple advancements being made within recent years. However, many aspects of this highly dynamic mechanism are still undefined. Some of these include (1) STIM resting state conformations, (2) how STIM transduces the gating signal to Orai after binding to its TM4x, and (3) how Orai relays that signal throughout the TM helices to the channel pore, which ultimately leads to its activation. Prior mutational studies have highlighted specific Leu residues on Orai’s TM4x that are necessary for Orai-STIM coupling, however, the residues on SOAR involved in this interaction are still undefined. This work details precise residues in the apical region of SOAR that play a major role in binding and tranducing the gating signal to Orai. We also identify two unusual overlapping Phe-His aromatic pairs within the STIM1 apical helix, one of which (F394-H398) is the crucial Orai1-coupling locus. Phe-394 is pivotal for binding Orai1, but His-398 is indispensable for transducing STIM1-binding into Orai1 channel-gating and is spatially aligned with Phe-394 in the exposed Sα2 helical apex. Additionally, artificial or naturally occurring gain of function (GoF) mutations act as a STIM-independent method for Orai activation, and further assist in solving the intramolecular Orai mechanisms that lead to channel gating. Multiple reports have used Orai1 GoF mutations to decipher structural components and resting/ active channel conformations, leading to speculation that Orai1 channel gating is a result of a global conformational change with multiple gating checkpoints. Therefore, this work also introduces three widely known Orai1 gain of function mutations to the other Orai isoforms as a STIM-independent method to further understand channel gating and signal transduction. Our results reveal unexpected but profound differences across the three Orai subtypes when mutating a critical Pro residue on the TM4, as well as important TM4-TM3 interactions in Orai2 that are responsible for the lack of constitutive activity compared to the other subtypes.