An essential and NSF independent role for α-SNAP in store-operated calcium entry

Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca2+ sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE.

Download Full-text

Bidirectional regulation of calcium release–activated calcium (CRAC) channel by SARAF

The Journal of Cell Biology ◽

10.1083/jcb.202104007 ◽

2021 ◽

Vol 220 (12) ◽

Author(s):

Elia Zomot ◽

Hadas Achildiev Cohen ◽

Inbal Dagan ◽

Ruslana Militsin ◽

Raz Palty

Keyword(s):

Gene Expression ◽

Calcium Release ◽

Cell Receptor ◽

Crac Channels ◽

Bidirectional Regulation ◽

Dependent Inactivation ◽

Store Operated Calcium Entry ◽

Crac Channel ◽

Initial Activation ◽

Downstream Gene Expression

Store-operated calcium entry (SOCE) through the Ca2+ release–activated Ca2+ (CRAC) channel is a central mechanism by which cells generate Ca2+ signals and mediate Ca2+-dependent gene expression. The molecular basis for CRAC channel regulation by the SOCE-associated regulatory factor (SARAF) remained insufficiently understood. Here we found that following ER Ca2+ depletion, SARAF facilitates a conformational change in the ER Ca2+ sensor STIM1 that relieves an activation constraint enforced by the STIM1 inactivation domain (ID; aa 475–483) and promotes initial activation of STIM1, its translocation to ER–plasma membrane junctions, and coupling to Orai1 channels. Following intracellular Ca2+ rise, cooperation between SARAF and the STIM1 ID controls CRAC channel slow Ca2+-dependent inactivation. We further show that in T lymphocytes, SARAF is required for proper T cell receptor evoked transcription. Taking all these data together, we uncover a dual regulatory role for SARAF during both activation and inactivation of CRAC channels and show that SARAF fine-tunes intracellular Ca2+ responses and downstream gene expression in cells.

Download Full-text

Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

10.1101/2020.04.04.025262 ◽

2020 ◽

Author(s):

Adam G Grieve ◽

Yi-Chun Yeh ◽

Lucrezia Zarcone ◽

Johannes Breuning ◽

Nicholas Johnson ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Calcium Release ◽

Calcium Influx ◽

Cell Mediated Immunity ◽

Crac Channels ◽

Rhomboid Proteases ◽

Store Operated Calcium Entry ◽

Crac Channel

SummaryCalcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease, RHBDL2. We show that RHBDL2 prevents stochastic signalling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved, disease-linked proline residue is responsible for RHBDL2 recognising only the active conformation of Orai1, and cleavage by RHBDL2 is required to sharpen switch-like signalling triggered by store-operated calcium entry. Loss of RHBDL2 control of Orai1 causes severe dysregulation of CRAC channel effectors including transcription factor activation, inflammatory cytokine expression and T cell activation. We propose that this seek-and-destroy function may represent an ancient activity of rhomboid proteases in degrading unwanted signalling proteins.

Download Full-text

Essential Role of Store-Operated Calcium Entry in Urotensin-Ii Induced Vascular Smooth Muscle Cells Proliferation

Biophysical Journal ◽

10.1016/j.bpj.2010.12.660 ◽

2011 ◽

Vol 100 (3) ◽

pp. 83a

Author(s):

María Rodríguez-Moyano ◽

Ignacio Díaz-Carrasco ◽

Alejandro Dominguez-Rodriguez ◽

Eva Calderon-Sanchez ◽

Antonio Ordoñez ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Essential Role ◽

Muscle Cells ◽

Calcium Entry ◽

Urotensin Ii ◽

Store Operated Calcium Entry

Download Full-text

Store-operated calcium entry and increased endothelial cell permeability

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.279.5.l815 ◽

2000 ◽

Vol 279 (5) ◽

pp. L815-L824 ◽

Cited By ~ 46

Author(s):

Natalie Norwood ◽

Timothy M. Moore ◽

David A. Dean ◽

Rakesh Bhattacharjee ◽

Ming Li ◽

...

Keyword(s):

Endothelial Cell ◽

Calcium Current ◽

Calcium Release ◽

Calcium Entry ◽

Extracellular Calcium ◽

Cell Permeability ◽

Calcium Stores ◽

Store Depletion ◽

Endothelial Cell Permeability ◽

Store Operated Calcium Entry

We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.

Download Full-text

A CRITICAL ROLE OF TRPC1-ORAI1-STIM1 MEDIATED STORE OPERATED CALCIUM ENTRY IN CARDIAC HYPERTROPHY

Journal of the American College of Cardiology ◽

10.1016/s0735-1097(15)60902-0 ◽

2015 ◽

Vol 65 (10) ◽

pp. A902

Author(s):

Senthil Selvaraj ◽

Brij Singh ◽

Christian Bollensdorff ◽

Jassim Al Suwaidi ◽

Magdi Yacoub

Keyword(s):

Cardiac Hypertrophy ◽

Critical Role ◽

Calcium Entry ◽

Store Operated Calcium Entry

Download Full-text

The Impact of Vitamin D3Supplementation on Mechanisms of Cell Calcium Signaling in Chronic Kidney Disease

BioMed Research International ◽

10.1155/2015/807673 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 1

Author(s):

Ingrid Lajdova ◽

Viera Spustova ◽

Adrian Oksa ◽

Zuzana Kaderjakova ◽

Dusan Chorvat ◽

...

Keyword(s):

Chronic Kidney Disease ◽

Kidney Disease ◽

Calcium Signaling ◽

Calcium Homeostasis ◽

Calcium Release ◽

Calcium Entry ◽

Regulatory Mechanisms ◽

Cell Calcium ◽

Crac Channels ◽

The Impact

Intracellular calcium concentration in peripheral blood mononuclear cells (PBMCs) of patients with chronic kidney disease (CKD) is significantly increased, and the regulatory mechanisms maintaining cellular calcium homeostasis are impaired. The purpose of this study was to examine the effect of vitaminD3on predominant regulatory mechanisms of cell calcium homeostasis. The study involved 16 CKD stages 2-3 patients with vitamin D deficiency treated with cholecalciferol 7000–14000 IU/week for 6 months. The regulatory mechanisms of calcium signaling were studied in PBMCs and red blood cells. After vitaminD3supplementation, serum concentration of 25(OH)D3increased (P<0.001) and[Ca2+]idecreased (P<0.001). The differences in[Ca2+]iwere inversely related to differences in 25(OH)D3concentration (P<0.01). VitaminD3supplementation decreased the calcium entry through calcium release activated calcium (CRAC) channels and purinergic P2X7channels. The function of P2X7receptors was changed in comparison with their baseline status, and the expression of these receptors was reduced. There was no effect of vitaminD3on P2X7pores and activity of plasma membrane Ca2+-ATPases. VitaminD3supplementation had a beneficial effect on[Ca2+]idecreasing calcium entry via CRAC and P2X7channels and reducing P2X7receptors expression.

Download Full-text