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A Calcium Influx Pathway Regulated Separately by Oxidative Stress and ADP-Ribose in TRPM2 Channels: Single Channel Events

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Abstract

A melastatin-like transient receptor potential 2 (TRPM2) channel is activated in concert with Ca2+ by intracellular adenosine diphosphoribose (ADPR) binding to the channel’s enzyme Nudix domain. Channel activity is also seen with nicotinamide dinucleotide (NAD+) and hydrogen peroxide (H2O2) although the mechanisms remain unknown. Hence, we tested the effects of ADPR, NAD+ and H2O2 on the activation of TRPM2 currents in transfected Chinese hamster ovary (CHO) cells. The CHO cells were transfected with cDNA coding for TRPM2. The intracellular solution used EDTA (10 mM) as a chelator for Ca2+ and heavy metal ions. Moreover, we balanced the intracellular Ca2+ concentration at 1 μM. H2O2 (10 mM) in the bath chamber was extracellularly added although ADPR (0.3 mM) and NAD+ (1 mM) in pipette solution were intracellularly added. Using these conditions, the channel currents were evoked by the three stimulators. The time course of ADPR, NAD+ and H2O2 effects was characterized by a delay of 0.6, 3.0 min and 2–5 min, respectively and a slow current induction reached a clear plateau with ADPR and NAD+ although H2O2 currents continued to gain in amplitude over at least 15 min and it did not reach a clear plateau in many experiments. Furthermore, H2O2-induced a single-channel conductance in the current study; the first time that this has been resolved in CHO. The conductance of ADPR and H2O2 was 48.80 pS and 39.14 pS, respectively and the cells seem to be separately activated by ADPR and H2O2. In conclusion, we observed further support for a calcium influx pathway regulated separately by oxidative stress and ADPR in TRPM2 channels in transfected cells. A second novel result of the present study was that the TRPM2 channels were constitutionally activated by H2O2.

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Abbreviations

ADPR:

Adenosine diphosphoribose

CHO:

Chinese hamster ovary

CRG-G1:

Cambridge rat insulinoma G1

HEK:

Human embryonic kidney

NAD+ :

Nicotinamide dinucleotide

NMDG:

N-methyl-D-glucamine

PARG:

Poly (ADP-ribose) glycohydrolase

PARP-1:

Poly (ADP-ribose) polymerase

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

TRP:

Transient receptor potential

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Acknowledgment

MN was supported for the study by an Alexander Humboldt Foundation (AvH) Research Fellowship. We thank Ilinca Ionescu and Eberhard Jungling for expert technical assistance and Dr. Peter J. Butterworth (King’s College, London) for their comments.

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Authors and Affiliations

  1. Department of Biophysics, Medical Faculty, Süleyman Demirel University, Cünür, Isparta, 32260, Turkey

    Mustafa Nazıroğlu

  2. Institute of Physiology, Medical Faculty, Rheinisch-Westfalische Technische Hochschule (RWTH), Aachen, Germany

    Andreas Lückhoff

Authors
  1. Mustafa Nazıroğlu
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  2. Andreas Lückhoff
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Corresponding author

Correspondence to Mustafa Nazıroğlu.

Additional information

A part of the study was submitted at the 1st Ion channels and Oxidative Stress Congress, Isparta, Turkey, 14–16 September, 2006.

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Nazıroğlu, M., Lückhoff, A. A Calcium Influx Pathway Regulated Separately by Oxidative Stress and ADP-Ribose in TRPM2 Channels: Single Channel Events. Neurochem Res 33, 1256–1262 (2008). https://doi.org/10.1007/s11064-007-9577-5

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  • DOI: https://doi.org/10.1007/s11064-007-9577-5

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