Amplification and propagation of pacemaker Ca2+ signals by cyclic ADP-ribose and the type 3 ryanodine receptor in T cells

Ligation of the T-cell receptor/CD3 complex results in global Ca2+ signals that are essential for T-cell activation. We have recently reported that these global Ca2+ signals are preceded by localized pacemaker Ca2+ signals. Here, we demonstrate for the first time for human T cells that an increase in signal frequency of subcellular pacemaker Ca2+ signals at sites close to the plasma membrane, in the cytosol and in the nucleus depends on the type 3 ryanodine receptor (RyR) and its modulation by cyclic ADP-ribose. The spatial distribution of D-myo-inositol 1,4,5-trisphosphate receptors and RyRs indicates a concerted action of both of these receptors/Ca2+ channels in the generation of initial pacemaker signals localized close to the plasma membrane. Inhibition or knockdown of RyRs resulted in significant decreases in (1) the frequency of initial pacemaker signals localized close to the plasma membrane, and (2) the frequency of localized pacemaker Ca2+ signals in the inner cytosol. Moreover, upon microinjection of cyclic ADP-ribose or upon extracellular addition of its novel membrane-permeant mimic N-1-ethoxymethyl-substituted cyclic inosine diphosphoribose, similarly decreased Ca2+ signals were observed in both type 3 RyR-knockdown cells and in control cells microinjected with the RyR antagonist Ruthenium Red. Taken together, our results show that, under physiological conditions in human T cells, RyRs play crucial roles in the local amplification and the spatiotemporal development of subcellular Ca2+ pacemaker signals.