UNDERSTANDING THE MECHANISM OF UNIMOLECULAR COUPLING BETWEEN STIM AND ORAI

Open Access
Author:
Nwokonko, Robert
Graduate Program:
Biomedical Sciences
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
August 29, 2019
Committee Members:
  • Donald Leon Gill, Dissertation Advisor/Co-Advisor
  • Mohamed Trebak, Committee Chair/Co-Chair
  • Thomas E Spratt, Committee Member
  • Blaise Peterson, Committee Member
  • Jong Kak Yun, Outside Member
  • Ralph Lauren Keil, Program Head/Chair
Keywords:
  • Store operated calcium channels
  • Electrophysiology
  • Membrane proteins
  • Orai calcium channel
  • Endoplasmic reticulum
  • Calcium signalling
Abstract:
Store-operated calcium (Ca2+) entry (SOCE) is a ubiquitous signaling mechanism in eukaryotic cells crucial for mediating longer–term Ca2+ signals and restoring endoplasmic/sarcoplasmic (ER/SR) reticulum Ca2+ after ligand induced depletion. The key operators in SOCE are the Ca2+ selective plasma membrane (PM) Orai1-3 channels and the ER/SR resident, single pass transmembrane Ca2+ sensors STIM1 and STIM2. STIM1 is activated when ER/SR luminal Ca2+ is depleted, inducing it to unfold and bind to Orai1 channels in the PM. Active Orai1 channels create discrete microdomains of high Ca2+ within ER-PM junctions that contain roughly 100-fold greater Ca2+ concentrations than resting cytoplasmic levels. Clustering of Orai channels is critical for generating Ca2+ saturated microdomains, however the mechanism of clustering is not well understood. We have discovered that Orai1 clustering is dependent on the presence of at least two functional C-terminal binding domains of the STIM/Orai Activating Region (SOAR) concatemer dimers. In HEK cells stably expressing Orai1 labelled with a repetitive histidine tag, Orai1-His cells, and transiently expressing wildtype SOAR-SOAR (S-S) concatemers, we observe an increase in SOAR-Orai1 colocalization, visualized as the formation of puncta in the plasma membrane. When one subunit within a SOAR concatemer has the phenylalanine-394 residue mutated to histidine (F394H) in either SH-S or S-SH configurations, a residue critical for high-affinity C-terminal binding to Orai1, there is a dramatic decrease in puncta. Similar to the F394H mutation, the STIM2 splice variant STIM2.1 has an additional 8 amino acid insertion within the C-terminal binding domain of the SOAR2 region, and concatemerized S-S2.1 peptides are also unable to cluster Orai1 channels. We also observe that linking a large fluorescent protein, CFP, to the C-terminus of Orai1 can sterically hinder clustering. HEK Orai1-CFP stable cells do not form puncta, regardless of the presence of two functional C-terminal binding domains on SOAR concatemers. There is also a functional difference in the calcium current passing through Orai channels (ICRAC) magnitude in HEK Orai1-His cells that is dependent on the presence of two functional SOAR subunits. Intriguingly, this dependence is absent in sterically hindered cells. Along with these biophysical studies, much has been done to study the role of Orai1 clustering in maintaining intracellular Ca2+ homeostasis. We find that the coexpression of full-length STIM1 and STIM2.1 dramatically impacts the ability for cells to maintain muscarinic agonist-induced regenerative Ca2+ oscillations in cells. My data suggests that this diminishment in oscillations is driven by decreased ER Ca2+ refilling in cells that are unable to cluster Orai1. We also observe that the ability for different NFAT isoforms to translocate into the nucleus after SOCE is altered by the ability for Orai1 to cluster or not cluster. In summary, my thesis research demonstrates that clustering of Orai1 channels is dependent on the presence of two functional SOAR subunits, and supports a unimolecular coupling mechanism between Orai and STIM that promotes full channel activity and the generation of Ca2+ saturated microdomains at ER-PM junctions that facilitates ER Ca2+ refilling and maintenance of prolonged intracellular Ca2+ signals. This suggests that eukaryotes may have evolved a mechanism mediated through regulation of STIM2 splicing that can affect the clustering dynamics of Orai ER-PM junctions in cells. However, much work on the regulation of STIM2.1 expression has yet to be done to understand how cells utilize it to affect the regulation of Ca2+ in vivo.