TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase.
1. TRPM2 (melastatin-like transient receptor potential 2 channel) is a nonselective cation channel that is activated under conditions of oxidative stress leading to an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and cell death. We investigated the role of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on hydrogen peroxide (H(2)O(2))-mediated TRPM2 activation using a tetracycline-inducible TRPM2-expressing cell line. 2. In whole-cell patch-clamp recordings, intracellular adenine 5'-diphosphoribose (ADP-ribose) triggered an inward current in tetracycline-induced TRPM2-human embryonic kidney (HEK293) cells, but not in uninduced cells. Similarly, H(2)O(2) stimulated an increase in [Ca(2+)](i) (pEC(50) 4.54+/-0.02) in Fluo-4-loaded TRPM2-expressing HEK293 cells, but not in uninduced cells. Induction of TRPM2 expression caused an increase in susceptibility to plasma membrane damage and mitochondrial dysfunction in response to H(2)O(2). These data demonstrate functional expression of TRPM2 following tetracycline induction in TRPM2-HEK293 cells. 3. PARP inhibitors SB750139-B (patent number DE10039610-A1 (Lubisch et al., 2001)), PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide) and DPQ (3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone) inhibited H(2)O(2)-mediated increases in [Ca(2+)](i) (pIC(50) vs 100 microm H(2)O(2): 7.64+/-0.38; 6.68+/-0.28; 4.78+/-0.05, respectively), increases in mitochondrial dysfunction (pIC(50) vs 300 microm H(2)O(2): 7.32+/-0.23; 6.69+/-0.22; 5.44+/-0.09, respectively) and decreases in plasma membrane integrity (pIC(50) vs 300 microm H(2)O(2): 7.45+/-0.27; 6.35+/-0.18; 5.29+/-0.12, respectively). The order of potency of the PARP inhibitors in these assays (SB750139>PJ34>DPQ) was the same as for inhibition of isolated PARP enzyme. 4. SB750139-B, PJ34 and DPQ had no effect on inward currents elicited by intracellular ADP-ribose in tetracycline-induced TRPM2-HEK293 cells, suggesting that PARP inhibitors are not interacting directly with the channel. 5. SB750139-B, PJ34 and DPQ inhibited increases in [Ca(2+)](i) in a rat insulinoma cell line (CRI-G1 cells) endogenously expressing TRPM2 (pIC(50) vs 100 microm H(2)O(2): 7.64+/-0.38; 6.68+/-0.28; 4.78+/-0.05, respectively). 6. These data suggest that oxidative stress causes TRPM2 channel opening in both recombinant and endogenously expressing cell systems via activation of PARP enzymes.
Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research and Development Limited, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW.
Elena
E
Fonfria
Ian C B
IC
Marshall
Christopher D
CD
Benham
Izzy
I
Boyfield
Jason D
JD
Brown
Kerstin
K
Hill
Jane P
JP
Hughes
Stephen D
SD
Skaper
Shaun
S
McNulty
D000246:Adenosine Diphosphate Ribose / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects; D004594:Electrophysiology; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D005470:Fluorometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007340:Insulinoma / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000187:drug effects*; D008565:Membrane Proteins / Q000187:drug effects*; D009930:Organic Chemicals; D018384:Oxidative Stress / Q000502:physiology*; D010190:Pancreatic Neoplasms / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D051381:Rats; D050053:TRPM Cation Channels; D013778:Tetrazolium Salts; D013844:Thiazoles
D005456:Fluorescent Dyes; D007473:Ion Channels; D008565:Membrane Proteins; D009930:Organic Chemicals; C402795:SYTOX Green; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D013778:Tetrazolium Salts; D013844:Thiazoles; D000246:Adenosine Diphosphate Ribose; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; C022616:thiazolyl blue; D002118:Calcium