Functional Ryanodine Receptor Expression Is Required for NAADP-mediated Local Ca2+ Signaling in T-lymphocytes

Werner Dammermann,Andreas H Guse

doi:10.1074/jbc.m413085200

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing nucleotide involved in T cell Ca2+ signaling (Berg, I., Potter, B. V. L., Mayr, G. W., and Guse, A. H. (2000) J. Cell Biol. 150, 581-588). The objective of this study was to analyze whether the first subcellular Ca2+ signals obtained upon NAADP stimulation of T-lymphocytes depend on the functional expression of ryanodine receptors. Using combined microinjection and high resolution confocal calcium imaging, we demonstrate here that subcellular Ca2+ signals, characterized by amplitudes between approximately 30 and 100 nM and diameters of approximately 0.5 microM, preceded global Ca2+ signals. Co-injection of the ryanodine receptor antagonists ruthenium red and ryanodine together with NAADP abolished the effects of NAADP, whereas the D-myo-inositol 1,4,5-trisphosphate antagonist heparin and the Ca2+ entry blocker SKF&96365 were without effect. This pharmacological approach was confirmed by a molecular knock-down approach. Jurkat T cell clones with largely reduced expression of ryanodine receptors did not respond to microinjections of NAADP. Taken together, our data suggest that the Ca2+ release channel sensitive to NAADP in T-lymphocytes is the ryanodine receptor.

Highlights

Nicotinic acid adenine dinucleotide phosphate (NAADP) 1 is an endogenous nucleotide in eukaryotic cells and to date represents the most powerful Ca2ϩ-releasing compound
We report that the initial subcellular Ca2ϩ signals induced by microinjection of NAADP depend on the functional expression of ryanodine receptor (RyR) in human T cells
In a recent report [13], we demonstrated that both RyR inhibition and the down-regulation of its expression almost completely abolished NAADP-mediated global Ca2ϩ signaling in T cells

Summary

Introduction

Nicotinic acid adenine dinucleotide phosphate (NAADP) 1 is an endogenous nucleotide in eukaryotic cells and to date represents the most powerful Ca2ϩ-releasing compound. A NAADP-sensitive Ca2ϩ pool was separated from the cADPR- and InsP3-sensitive one by stratification of sea urchin eggs [5] This store was identified as the reserve granule of eggs, a lysosome-related organelle [6]. The pharmacological characterization of NAADPinduced Ca2ϩ signaling in sea urchin eggs resulted in the conclusion that a novel Ca2ϩ channel unrelated to the known intracellular Ca2ϩ release channels is involved, a number of reports from heart and skeletal muscle and pancreatic acinar cells suggest that RyR are the Ca2ϩ channels mediating the effect of NAADP (9 –11). Because that report was compatible with both models, the present study was conducted to analyze the very initial subcellular Ca2ϩ release events observed upon NAADP stimulation and, in particular, to understand whether the RyR is necessary for these spatiotemporally restricted Ca2ϩ signals

Objectives

Results

Conclusion