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.

NAADP-evoked Ca2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1–like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains and delayed the onset and decreased the amplitude of global Ca2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3–dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through RYR.

The enzymes DUOX1 and DUOX2 may mediate rapid and then sustained NAADP production to promote Ca 2+ signaling in T cells.