ORAI1, STIM1/2, and RYR1 shape subsecond Ca ²⁺ microdomains upon T cell activation

Abstract

The earliest intracellular signals determined in T cell activation are local, sub-second Ca2+ microdomains (1). Here we identify a Ca2+ entry component involved in Ca2+ microdomain formation in both non-stimulated and stimulated cells. In non-stimulated cells, spontaneous small Ca2+ microdomains depend on expression of ORAI1, STIM1, and STIM2. Using T cells stably transfected with ORAI1 fused to a genetically encoded Ca2+ indicator for optical imaging spontaneous Ca2+ microdomains depending on ORAI1 were also detected. Super resolution microscopy of non-stimulated T cells resulted in identification of a circular subplasmalemmal region with a diameter of approx. 300 nm with preformed patches of co-localized ORAI1, ryanodine receptors (RYR), and STIM1. Preformed complexes of STIM1 and ORAI1 in non-stimulated cells were confirmed by co-immunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second of T cell receptor (TCR) stimulation, Ca2+ microdomain numbers increase in the subplasmalemmal space, an effect not observed upon genetic deletion of Orai1, Stim2 or Ryr1 or upon antagonism of the Ca2+ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Taken together, while preformed clusters of STIM and ORAI1 allow for local Ca2+ entry events in non-stimulated cells, upon TCR activation, NAADP-evoked Ca2+ release via RYR1, in tight interplay with Ca2+ entry via ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.

Electrostatic interactions: From immune receptor assembly to signaling

“…The cytosolic calcium concentration in resting T cells is maintained at 100 nm, while extracellular calcium is around 2 mM. Fast imaging studies of T cells activated with anti‐CD3‐coated beads or peptide‐pulsed APCs have shown that calcium microdomains are formed at the synapse within 1 second after T cell‐bead contact, while overall increase in cytosolic calcium was observed within seconds following synapse initiation …”

Periodic Membrane Potential and Ca2+ Oscillations in T Cells Forming an Immune Synapse

“…This way Ca 2+ influx through a Ca 2+ channel associated with a BK channel can be sufficient to significantly shift the voltage-dependence of BK and activate the channel in the complex. Recently, the significance of channel complexes and Ca 2+ microdomains during early T cell activation was also demonstrated using super-resolution microscopy [48].…”

25 Years of Collaboration with A Genius: Deciphering Adenine Nucleotide Ca2+ Mobilizing Second Messengers Together with Professor Barry Potter

“…Ca 2+ signaling can be further subdivided into NAADP- and ORAI1-dependent local Ca 2+ signals [ 7 , 8 ], IP 3 -dependent global Ca 2+ signaling, and cADPR and ryanodine receptor (RYR)-dependent global Ca 2+ signaling ( Figure 1 ). In the following sections, I will first concentrate on NAADP and cADPR as two of the three main bonds between Barry Potter’s lab and my group.…”

“…The first signals that can be monitored during T cell activation are local, short-lived and highly dynamic Ca 2+ signals, termed Ca 2+ microdomains [ 7 , 8 ]. Besides gene silencing approaches to disentangle the molecular mechanisms involved, an analogue of NAADP synthesized initially by Barry Potter’s group was used, namely BZ194 [ 9 ].…”

“…One of the hits, BZ194, was then validated by microinjection experiments as above and resulted in an IC 50 of approximately 5 μM towards NAADP evoked Ca 2+ signaling; furthermore, when BZ194 was co-injected with IP 3 or cADPR, no inhibition was observed, demonstrating the specificity of this NAADP antagonist [ 9 ]. BZ194 significantly decreases the number of Ca 2+ microdomains within the first 15 s of T cell activation in primary T cells [ 8 ]. Mechanistically, BZ194 interferes with RYR1 opening, as shown in [ 3 H]ryanodine binding assays [ 9 ].…”