Cyclic ADP-Ribose and Hydrogen Peroxide Synergize with ADP-Ribose in the Activation of TRPM2 Channels

Abstract

The melastatin-related transient receptor potential channel TRPM2 is a plasma membrane Ca2+-permeable cation channel that is activated by intracellular adenosine diphosphoribose (ADPR) binding to the channel's enzymatic Nudix domain. Channel activity is also seen with nicotinamide dinucleotide (NAD+) and hydrogen peroxide (H2O2), but their mechanisms of action remain unknown. Here, we identify cyclic adenosine diphosphoribose (cADPR) as an agonist of TRPM2 with dual activity: at concentrations above 100 microM, cADPR can gate the channel by itself, whereas lower concentrations of 10 microM have a potentiating effect that enables ADPR to gate the channel at nanomolar concentrations. ADPR's breakdown product adenosine monophosphate (AMP) specifically inhibits ADPR, but not cADPR-mediated gating of TRPM2, whereas the cADPR antagonist 8-Br-cADPR exhibits the reverse block specificity. Our results establish TRPM2 as a coincidence detector for ADPR and cADPR signaling and provide a functional context for cADPR as a second messenger for Ca2+ influx.

Synergistic regulation of endogenous TRPM2 channels by adenine dinucleotides in primary human neutrophils

“…A doseresponse fit to the data yielded a half-maximal effective concentration (EC 50 ) for ADPR of 1.1 μM with a Hill coefficient of 1.5. This is considerably lower than the EC 50 values obtained for heterologously expressed TRPM2 in HEK293 cells (10 μM) [7,16], or native TRPM2 in Jurkat T cells (7 μM) [10], U937 monocytes (40 μM) [4], and RINm5f cells (20 μM, Fleig unpublished observations), indicating that both ionic and cellular environment can determine the sensitivity of TRPM2 channels to ADPR. Several cellular systems have been shown to require the presence of intracellular and/or extracellular Ca 2+ to evoke ADPR-induced TRPM2 currents, including human neutrophils [4,12,[14][15][16].…”

“…We have previously reported that cADPR, H 2 O 2 , and NAADP can synergize with the primary agonist ADPR to more efficiently activate TRPM2 [7] and we recently demonstrated in Jurkat T cells and HEK293 cells overexpressing TRPM2 that intracellular cations can affect the sensitivity of TRPM2 channels to ADPR and cADPR [7,10], which might also affect the synergistic effects of other TRPM2 modulators. Previous studies on TRPM2 in primary neutrophils employed Cs + -based intracellular solutions [12,20], and one study reported the failure of cADPR to activate or synergize with ADPR and AMP to inhibit ADPR-induced TRPM2 currents in human neutrophils [12].…”

“…ADPR-induced activation of TRPM2 currents are negatively regulated by increasing intracellular AMP concentrations in Jurkat T cells and HEK293 cells overexpressing the channel [7,10]. However, lower concentrations of AMP (5 μM) reportedly failed to inhibit TRPM2 in neutrophils activated by saturating ADPR concentrations (5 μM) in intracellular Cs + conditions [12].…”

“…The primary channelgating mechanism is mediated by ADPR [4,7], although limited activation of TRPM2 can be accomplished by H 2 O 2 [2,8,9] and relatively high concentrations of cyclic ADP-ribose (cADPR) [7,10,11]. However, cADPR, and H 2 O 2 can synergize with ADPR to activate TRPM2 currents at ADPR levels in the low nanomolar range [7], although one report has claimed that this does not seem to be the case for human neutrophils [12]. Another activator of TRPM2 channels in overexpressing HEK293 cells and Jurkat T lymphocytes is the highly efficient Ca 2+ -release agent nicotinic acid adenine dinucleotide phosphate (NAADP, [13]), which can also facilitate ADPR action on TRPM2 [10].…”

Selective inhibition of TRPM2 channel by two novel synthesized ADPR analogues

“…In addition, several nucleotide analogues such as adenosine monophosphate (AMP), 8‐bromoadenosine 5′‐diphosphoribose (8‐Br‐ADPR) have also been reported to be TRPM2 inhibitors (Figure 1). 26, 27 Recently, a series of ADPR analogues mainly modified at C‐8 position of purine such as 8‐phenyl‐2′‐ deoxy ‐ADPR (Figure 1) have been shown to potently inhibit TRPM2 current (IC 50  = 3 μ m ) without interfering Ca 2+ release induced by cADPR, NAADP or IP 3 28. Despite the efforts in searching for TRPM2 inhibitors, no TRP‐subtype selective TRPM2 inhibitor has yet been identified.…”