Search

Initial T cell activation is triggered by the formation of highly dynamic, spatiotemporally restricted Ca2+microdomains. Purinergic signaling is known to be involved in Ca2+influx in T cells at later stages compared to the initial microdomain formation. Using a high-resolution Ca2+live-cell imaging system, we show that the two purinergic cation channels P2X4 and P2X7 not only are involved in the global Ca2+signals but also promote initial Ca2+microdomains tens of milliseconds after T cell stimulation. These Ca2+microdomains were significantly decreased in T cells fromP2rx4−/−andP2rx7−/−mice or by pharmacological inhibition or blocking. Furthermore, we show a pannexin-1–dependent activation of P2X4 in the absence of T cell receptor/CD3 stimulation. Subsequently, upon T cell receptor/CD3 stimulation, ATP release is increased and autocrine activation of both P2X4 and P2X7 then amplifies initial Ca2+microdomains already in the first second of 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.

Intracellular calcium (Ca2+) signals are of prime importance for cellular function and behavior and are underpinned by a plethora of Ca2+ channels, pumps, transporters, and binding proteins that are regulated in complex ways. A series of biennial meetings, the International Meetings of the European Calcium Society (ECS), focuses on a better understanding of these complex mechanisms in the framework of cellular and organismal (patho)physiology.

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca2+ microdomains. Here, we identified a Ca2+ entry component involved in Ca2+ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca2+ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Forster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca2+ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca2+ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca2+ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca2+ release through RYR1, in coordination with Ca2+ entry through 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.

Nicotinamide adenine dinucleotide (NAD(+)) is the universal currency of energy metabolism and electron transfer. Recent studies indicate that apart from its role as a coenzyme, NAD(+) and its metabolites also function in cell signaling pathways; for example, they are substrates for nucleotide-metabolizing enzymes and ligands for extra- and intracellular receptors and ion channels. Moreover, the NAD(+) and NAD(+) phosphate metabolites adenosine 5'-diphosphoribose (ADP-ribose), cyclic ADP-ribose, and nicotinic acid adenine dinucleotide phosphate (NAADP) have emerged as key second messengers in Ca(2+) signaling. A symposium in Hamburg, Germany, brought together 120 researchers from various fields, who were all engaged in the molecular characterization of the key players of NAD(+) signaling (www.NAD2008.de).

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca 2+ -mobilizing second messenger found in cells of animals, plants, and protozoans. It is formed by a specific class of enzymes, the ADP-ribosyl cyclases. cADPR stimulates Ca 2+ release by means of ryanodine receptors located in the sarcoplasmic and endoplasmic reticulum. Recently, a role for cADPR has been demonstrated in the obligate intracellular protozoan pathogen Toxoplasma gondii . In T. gondii , stress conditions evoked synthesis of the plant hormone abscisic acid by the apicoplast, a remnant organelle of an algal endosymbiont of T. gondii . Abscisic acid in turn activated formation of cADPR within T. gondii , resulting in Ca 2+ release and secretion of proteins involved in egress of T. gondii from its host cell. Evidence for a synthetic pathway of plant origin was obtained with the ABA synthesis inhibitor fluridone, which antagonized cellular egress and induced differentiation of long-lived semidormant cystic forms of T. gondii. Moreover, fluridone protected mice from toxoplasmosis.