Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes
Résumé
TRPM2 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.
Origine : Fichiers produits par l'(les) auteur(s)
Loading...