Abstract
Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel involved in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca(2+) and ADP-ribose (ADPR) but also modulated in intact cells by several additional factors. Superfusion of TRPM2-expressing cells with H(2)O(2) or intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) activates, whereas dialysis of AMP inhibits, TRPM2 whole-cell currents. Additionally, H(2)O(2), cADPR, and NAADP enhance ADPR sensitivity of TRPM2 currents in intact cells. Because in whole-cell recordings the entire cellular machinery for nucleotide and Ca(2+) homeostasis is intact, modulators might affect TRPM2 activity either directly, by binding to TRPM2, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca(2+). To identify direct modulators of TRPM2, we have studied the effects of H(2)O(2), AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide in inside-out patches from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H(2)O(2) (1 mM) and enzymatically purified cADPR (10 μM) failed to activate, whereas AMP (200 μM) failed to inhibit TRPM2 currents. NAADP was a partial agonist (maximal efficacy, ∼50%), and nicotinic acid adenine dinucleotide was a full agonist, but both had very low affinities (K(0.5) = 104 and 35 μM). H(2)O(2), cADPR, and NAADP did not enhance activation by ADPR. Considering intracellular concentrations of these compounds, none of them are likely to directly affect the TRPM2 channel protein in a physiological context.
Highlights
Ute to chemotactic responses (5) and chemokine production (6) of immune cells, as well as to neuronal cell death following ischemia (4, 7, 8)
F, in the presence of 1 M Ca2ϩ, sible for Transient receptor potential melastatin 2 (TRPM2) activation are found in a restricted space intracellularly, but in the immediate vicinity of the channel pore, such that the primary source for activating Ca2ϩ negligible fractional currents are activated even by 1000 M ADPR co-applied with 2 M calmodulin (CAM); the is likely extracellular
ADPR Fail to Activate TRPM2 Channels at Submicromolar [Ca2ϩ]—Because ADPR affinities reported in previous studies were measured in intact cells in which bulk cytosolic Ca2ϩ levels are submicromolar (2, 16, 18), we evaluated the possibility of measuring TRPM2 sensitivity to ADPR at submicromolar Ca2ϩ levels in our cell-free system
Summary
Molecular Biology— hTRPM2/pGEMHE was constructed as described (9). For in vitro transcription plasmid DNA was linearized by NheI; cRNA was prepared using a T7 mMessage mMachine kit (Ambion) and quantified on denaturing gels. Macroscopic current recordings were obtained 2–3 days after injection of 10 ng of hTRPM2 cRNA. Excised Patch Recordings—Inside-out patch recordings were done at 25 °C in a symmetrical sodium gluconate-based solution at a pipette holding potential of ϩ20 mV (Vm ϭ Ϫ20 mV) as described (9). Data Analysis—To obtain fractional activation by some test concentration of a given nucleotide, quasi-steady macroscopic TRPM2 current in the test segment was normalized to the maximal activity estimated for the same segment. Because after patch excision TRPM2 currents continuously decline (due to a progressive loss of active channels in the patch (9)), the maximal activity used for normalization was obtained by linear interpolation of the activities in the presence of saturating (32 M) ADPR, obtained in bracketing segments of record in the same patch. For test applications that preceded exposure to 32 M ADPR, maximal activity for the test segment was estimated by linear extrapolation of the activities obtained subsequently, in the same patch, upon repeated exposures to 32 M ADPR
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