TRPM2 (transient receptor potential melastatin 2) is a Ca2+-permeable cation channel gated by ADPR (ADP-ribose) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC method to determine ADPR contents in HClO4 cell extracts. The reversed-phase ion-pair HPLC method with an Mg2+-containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were approx. 5 μM in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP (N-formylmethionyl-leucylphenylalanine). We furthermore determined intracellular concentrations of cADPR (cyclic ADPR) with a cyclase assay involving enzymatic conversion of cADPR into NAD+ and fluorimetric determination of NAD+. Intracellular cADPR concentrations were approx. 0.2 μM and not altered by fMLP. In patch–clamp experiments, ADPR (0.1–100 μM) was dialysed into granulocytes to analyse its effects on whole-cell currents characteristic for TRPM2, in the presence of a low (<10 nM) or a high (1 μM) intracellular Ca2+ concentration. TRPM2 currents were significantly larger at high than at low [Ca2+] (e.g. −225±27.1 versus −7±2.0 pA/pF at 5 μM ADPR), but no currents at all were observed in the absence of ADPR (ADPR concentration ≤0.3 μM). cADPR (0.1, 0.3 and 10 μM) was without effect even in the presence of subthreshold ADPR (0.1 μM). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca2+. Thus ADPR and Ca2+ in concert behave as a messenger system for agonist-induced influx of Ca2+ through TRPM2 in granulocytes.
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Research Article|
August 15 2006
Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes
Inka Heiner;
Inka Heiner
1Medical Faculty, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstr. 30, D-52057 Aachen, Germany
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Jörg Eisfeld;
Jörg Eisfeld
1Medical Faculty, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstr. 30, D-52057 Aachen, Germany
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Maike Warnstedt;
Maike Warnstedt
1Medical Faculty, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstr. 30, D-52057 Aachen, Germany
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Natalia Radukina;
Natalia Radukina
1Medical Faculty, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstr. 30, D-52057 Aachen, Germany
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Eberhard Jüngling;
Eberhard Jüngling
1Medical Faculty, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstr. 30, D-52057 Aachen, Germany
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Andreas Lückhoff
Andreas Lückhoff
1
1Medical Faculty, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstr. 30, D-52057 Aachen, Germany
1To whom correspondence should be addressed (email luckhoff@physiology.rwth-aachen.de).
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Publisher: Portland Press Ltd
Received:
January 31 2006
Revision Received:
May 04 2006
Accepted:
May 09 2006
Accepted Manuscript online:
May 09 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 398 (2): 225–232.
Article history
Received:
January 31 2006
Revision Received:
May 04 2006
Accepted:
May 09 2006
Accepted Manuscript online:
May 09 2006
Citation
Inka Heiner, Jörg Eisfeld, Maike Warnstedt, Natalia Radukina, Eberhard Jüngling, Andreas Lückhoff; Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes. Biochem J 1 September 2006; 398 (2): 225–232. doi: https://doi.org/10.1042/BJ20060183
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