Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes

Abstract

International audienceTRPM2 is a Ca 2+}-permeable cation channel gated by ADP-ribose (ADPR) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC protocol to determine ADPR contents in perchloric acid cell extracts. The reversed phase ion-pair HPLC protocol with a Mg 2+} containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were about 5 {mu}M in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP. We furthermore analyzed intracellular concentrations of cyclic ADPR (cADPR) with a cyclase assay involving enzymatic conversion of cADPR to NAD and fluorometric determination of NAD. Intracellular cADPR concentrations were about 0.2 {mu}M and not altered by fMLP. In patch-clamp experiments, ADPR (0.1 to 100 {mu}M) was dialyzed into granulocytes to analyze its effects on whole-cell currents characteristic for TRPM2, in the presence of a low (< 10 nM) or a high (1 {mu}M) intracellular Ca 2+} concentration. TRPM2 currents were significantly larger in high than in low Ca 2+} (e.g. -225 ± 27.1 vs. -7 ± 2.0 pA/pF at 5 {mu}M ADPR) but no currents at all were observed in the absence of ADPR (ADPR concentration <= 0.3 {mu}M). cADPR (0.1, 0.3 and 10 {mu}M) was without effect even in the presence of subthreshold ADPR (0.1 {mu}M). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca 2+}. Thus, ADPR and Ca 2+} in concert behave as a messenger system for agonist-induced influx of Ca 2+} through TRPM2 in granulocytes

Synergistic regulation of endogenous TRPM2 channels by adenine dinucleotides in primary human neutrophils

“…Previous studies on TRPM2 in primary neutrophils employed Cs + -based intracellular solutions [12,20], and one study reported the failure of cADPR to activate or synergize with ADPR and AMP to inhibit ADPR-induced TRPM2 currents in human neutrophils [12]. Since these studies provided results under somewhat limited experimental conditions, we conducted a detailed analysis of the facilitatory actions of cADPR, H 2 O 2 and NAADP in relation to ADPR-induced TRPM2 activation in primary human neutrophils, using K + -based solutions and assessing agonist effects over a large concentration range.…”

“…This is considerably lower than the EC 50 values obtained for heterologously expressed TRPM2 in HEK293 cells (10 μM) [7,16], or native TRPM2 in Jurkat T cells (7 μM) [10], U937 monocytes (40 μM) [4], and RINm5f cells (20 μM, Fleig unpublished observations), indicating that both ionic and cellular environment can determine the sensitivity of TRPM2 channels to ADPR. Several cellular systems have been shown to require the presence of intracellular and/or extracellular Ca 2+ to evoke ADPR-induced TRPM2 currents, including human neutrophils [4,12,[14][15][16]. When perfusing human neutrophils with a fixed concentrations of 1 mM ADPR in the presence of increasing intracellular Ca 2+ concentrations ranging from 0 to 1 μM ( Fig.…”

“…Ca 2+ -dependence of TRPM2 activity has been reported in a variety of cell lines [4,12,[14][15][16]18], as well as in primary systems such as neutrophil granulocytes [12].…”

“…The primary channelgating mechanism is mediated by ADPR [4,7], although limited activation of TRPM2 can be accomplished by H 2 O 2 [2,8,9] and relatively high concentrations of cyclic ADP-ribose (cADPR) [7,10,11]. However, cADPR, and H 2 O 2 can synergize with ADPR to activate TRPM2 currents at ADPR levels in the low nanomolar range [7], although one report has claimed that this does not seem to be the case for human neutrophils [12]. Another activator of TRPM2 channels in overexpressing HEK293 cells and Jurkat T lymphocytes is the highly efficient Ca 2+ -release agent nicotinic acid adenine dinucleotide phosphate (NAADP, [13]), which can also facilitate ADPR action on TRPM2 [10].…”

“…Another activator of TRPM2 channels in overexpressing HEK293 cells and Jurkat T lymphocytes is the highly efficient Ca 2+ -release agent nicotinic acid adenine dinucleotide phosphate (NAADP, [13]), which can also facilitate ADPR action on TRPM2 [10]. At the same time, the channel's sensitivity to ADPR is potentiated by intracellular Ca 2+ [4,12,[14][15][16] acting as a positive feedback mechanism on channel activity, likely mediated by calmodulin [16,17]. Furthermore, *Correspondence should be addressed to: Andrea Fleig, PhD, 1301 Punchbowl Street, Honolulu, Hi 96813.…”