The Coordinated Functions of STIM1/2, ORAI1/2/3, and MCU Underlie the Diversity of Physiological Ca2+ Signaling
Open Access
- Author:
- Yoast, Ryan
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 16, 2021
- Committee Members:
- David Degraff, Outside Field Member
Mohamed Trebak, Chair & Dissertation Advisor
Todd Schell, Outside Unit Member
Christopher Yengo, Major Field Member
Ralph Keil, Program Head/Chair - Keywords:
- STIM
ORAI
MCU
Calcium Signaling
Calcium Oscillations
SOCE
CRAC channels
NFAT
Mitochondrial Calcium
IP3R
CRISPR/Cas9 - Abstract:
- Calcium ions (Ca2+) are ubiquitous and versatile members of the cellular signaling toolkit. In non-excitable cells, the store-operated Ca2+ entry (SOCE) pathway—activated in response to endoplasmic reticulum (ER) Ca2+ store depletion—is the predominant Ca2+ entry mechanism. The SOCE pathway consists of three plasma membrane (PM) resident channel proteins (ORAI1-3) and two ER-resident Ca2+ sensors (STIM1-2) that activate ORAI channels. While it is established that ORAI1 is an essential component of the native ORAI channel, the contributions of ORAI2 and ORAI3 to native SOCE were unclear. To determine the physiological role of ORAI2 and ORAI3, we genetically knocked out a single, pair, or all three ORAI isoforms in HEK293 cells. Doing so revealed that ORAI2 and ORAI3 were capable of mediating cytosolic Ca2+ oscillations while ORAI1 was dispensable. Further functional and biochemical analysis revealed that ORAI2 and ORAI3 can form heteromeric channels with ORAI1 to expand the bandwidth of Ca2+ signaling events. This broadened bandwidth physiologically manifested in differential NFAT isoform activation. Further investigation sought to determine the contribution of STIM proteins in shaping the native Ca2+ signal. Using a similar genetic approach, we showed that all five SOCE proteins STIM1/2 and ORAI1/2/3 play non-redundant roles in expanding Ca2+ signal diversity. Unactivated STIM molecules were found to negatively regulate the activity of ER-resident IP3Rs. Functional interactions between STIM1/2, ORAI1/2/3, and the IP3R shape the native Ca2+ signal and NFAT activation in response to a broad range of agonist concentrations. Specifically, we report that the activated C-termini of STIM1/2 differentially interact with all ORAI isoforms in a concentration-dependent manner. Thus, the omnitemporal functions of all SOCE and IP3R proteins culminate in the precise tuning of the Ca2+ signal to enhance the fidelity of transcriptional activation. Organelles, including the mitochondria, shape cytosolic Ca2+ signals; an observation that was largely attributed to direct regulation of CRAC channel activity by mitochondria. However, knockout of the predominant mitochondrial Ca2+ uptake channel―MCU—from multiple cell lines and primary mouse T and B cells revealed this was not the case. We show that MCU-KO increases the maximal Ca2+ signal in response to ER store depletion by acutely buffering cytosolic Ca2+, despite enhancing Ca2+ extrusion and promoting the slow Ca2+-dependent inactivation of CRAC channels. Knockout of MCU amplified the cytosolic Ca2+ signal increasing NFAT activation and the rate of primary B cell proliferation. This body of work represents a substantial leap forward in understanding how multiple PM and organellar Ca2+ channels and sensors shape the native Ca2+ signal and provides critical insights into how modulation of this signal influences physiology.