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Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes

Laboratory of Cell and Molecular Signaling, Center for Biomedical Research at The Queen’s Medical Center, and John A. Burns School of Medicine at the University of Hawaii, Honolulu, Hawaii, USA

²Correspondence: Center for Biomedical Research, The Queen’s Medical Center, 1301 Punchbowl St. UHT 8, Honolulu, HI 96813 USA. E-mail: rpenner{at}hawaii.edu

SPECIFIC AIMS

TRPM2 is a Ca²⁺-permeable non-selective cation channel that contains a C-terminal enzymatic domain with pyrophosphatase activity, which specifically binds ADP-ribose. Recent experiments have shown that cyclic ADP-ribose (cADPR) and hydrogen peroxide (H₂O₂) can facilitate ADPR-mediated activation of heterologously expressed TRPM2. This study aimed to determine whether two Ca²⁺-mobilizing second messengers, specifically cADPR and nicotinic acid adenine dinucleotide phosphate (NAADP), could activate natively expressed TRPM2 channels in Jurkat T cells and to test the hypothesis whether these two agonists share a common binding site on TRPM2 that can regulate TRPM2 activity in synergy with ADPR.

PRINCIPAL FINDINGS

1. ADPR and cADPR activate native TRPM2 currents in Jurkat T cells.
Northern blotting has indicated that TRPM2 is expressed in a variety of tissues, and ADPR has been shown to produce currents (I_ADPR) in monocyte and T lymphocyte cell lines. To determine the characteristics of the I_ADPR current in Jurkat T lymphocytes, we performed whole-cell experiments using both ADPR and cADPR as channel activators. Our data revealed a maximum whole-cell current of 500 pA at 1 mM internal ADPR (Fig. 1 A). A similar current size was reached at doses as low as 10 µM. cADPR induced maximum current sizes of around 600 pA (Fig. 1B ) but required slightly higher concentrations than ADPR and was effective at concentrations of 100 µM or higher. The linear current-voltage relationships of the currents produced by either agonist were indistinguishable from each other (Fig. 1C ). The dose-response relationships derived from these whole-cell currents are shown in Fig. 1D . The half-maximal effective concentration (EC₅₀) for cADPR was found to be 60 µM and that of ADPR was 10 µM. These data indicate that the I_ADPR channel can be gated by both ADPR and cADPR in lymphocytes, although these cells are more sensitive to ADPR as a gating mechanism than to cADPR.

View larger version (33K): [in this window] [in a new window]   Figure 1. Native TRPM2 currents in Jurkat T cells. A) Average membrane currents recorded at –80 mV induced by perfusion of Jurkat cells with various concentrations of ADPR ranging from 3 µM to 1 mM (n=5–8) (B) Average membrane currents recorded at –80 mV induced with various concentrations of cADPR ranging from 30 µM to 1 mM (n=4–5). C) Representative current-voltage relationships derived from currents evoked by voltage ramps spanning –100 to + 100 mV. D) Dose-response relationships for IADPR in Jurkat cells evoked by ADPR (filled symbols) or cADPR (open symbols).

To establish that ADPR and cADPR can directly affect the I_ADPR channel in Jurkat cells, we applied either of these agonists to the internal side of cell-free inside-out membrane patches. Individual channel openings were observed using both ADPR and cADPR (Fig. 1E ), although ADPR induced channel activity in only 11 out of 58 patches (19%), and cADPR was effective in 15 out of 111 patches (14%). The current-voltage relationships derived from these inside-out patches are shown in Fig. 1F and are virtually indistinguishable from each other. The single-channel conductance obtained by linear fits were 67 pS for ADPR and 69 pS for cADPR. These data indicate that the I_ADPR channel in Jurkat cells can be gated directly by either ADPR or cADPR in cell-free excised membrane patches.

2. ADPR- and cADPR-induced native TRPM2 currents in Jurkat cells have similar biophysical characteristics compared with heterologously expressed TRPM2 channels in HEK-293 cells.
The main difference we observed between heterologously expressed TRPM2 and the native I_ADPR in T cells was that the sensitivity of I_ADPR toward cADPR is significantly lower in T cells (EC₅₀=60 µM) compared with HEK-293 cells (EC₅₀=120 µM). In addition, the maximal current amplitudes obtained by cADPR in T cells are comparable with those achieved by ADPR, whereas our previous study found only a rather limited extent of cADPR-mediated activation. We considered that one possible reason for this discrepancy might reside in different intracellular pipette solutions used by the two studies, since the above T cell experiments were all performed using Cs-glutamate-based pipette solutions to minimize the lymphocyte’s endogenous K⁺ currents, whereas our previous study on heterologously expressed TRPM2 in HEK- 293 cells was performed with K-glutamate-based solutions.

We therefore reassessed the ADPR and cADPR effects in both T cells and HEK-293 cells using K- and Cs-glutamate-based pipette solutions. Both ADPR and cADPR dose-dependently activated I_ADPR currents in K⁺-based solutions. No significant change was found in the apparent sensitivity of TRPM2 to either ADPR or cADPR; the half-maximal effective concentrations for ADPR in the presence of Cs⁺ and K⁺ were 7 and 15 µM, respectively, and for cADPR they were 60 µM in both cases. The ADPR values are in close agreement to the EC₅₀ values we determined in HEK-293 cells in Cs⁺- and K⁺-based solutions (15 and 12 µM, respectively). In both cell types, the maximal ADPR-induced current amplitudes were slightly smaller in the presence of Cs⁺, suggesting that Cs⁺ may actually reduce channel open probability. In contrast, we observed significant differences in the behavior of TRPM2 in both cell types when stimulating with cADPR. In T cells, TRPM2’s sensitivity toward cADPR was unchanged, but current amplitudes were generally larger in the presence of Cs⁺. In HEK-293 cells, cADPR was not very effective in K⁺-based solutions, but Cs⁺ shifted the EC₅₀ by a factor of 6 from 700 µM in the presence of K⁺ to 120 µM in Cs⁺-based solutions. In addition, the maximal cADPR-induced current amplitudes were larger in the presence of Cs⁺. Thus, the overall sensitivity of the native channels in lymphocytes toward cADPR remains significantly higher than that of heterologously expressed TRPM2 in HEK-293 cells under either ionic condition.

3. NAADP activates TRPM2 in HEK-293 and Jurkat T cells.
More recently, a new Ca²⁺-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) has emerged. Like cADPR, NAADP can be produced via CD38 and like cADPR it appears to cause both Ca²⁺ release and Ca²⁺ influx, although no current consensus exists on whether NAADP targets the same receptor as cADPR. We considered the possibility that NAADP might affect TRPM2 channels and perfused HEK-293 cells expressing TRPM2 with various concentrations of this putative second messenger. NAADP indeed activated TRPM2 currents (Fig. 2 A) with a typical linear current-voltage relationship (Fig. 2B ) and in a dose-dependent manner with an EC₅₀ of 730 µM (Fig. 2C ). We confirmed that NAADP could also activate I_ADPR in Jurkat T cells (Fig. 2D ) and proceeded to characterize the NAADP mechanism in HEK-293 cells.

View larger version (14K): [in this window] [in a new window]   Figure 2. NAADP activates TRPM2 in HEK-293 and Jurkat T cells. A) Average membrane currents recorded at –80 mV induced by perfusion of TRPM2-expressing HEK-293 cells with various concentrations of NAADP ranging from 30 µM to 3 mM (n=4–7). B) Representative current-voltage relationship derived from currents evoked by voltage ramps spanning –100 to + 100 mV. C) Average membrane currents recorded at –80 and + 80 mV induced by perfusion of Jurkat T cells with 1 mM NAADP (n=5).

4. NAADP-induced TRPM2 currents are inhibited by both AMP and 8-Br-cADPR.
Since the efficacy of NAADP in activating TRPM2 was similar to that of cADPR, we reasoned that it might also synergize with ADPR at significantly lower concentrations. Indeed, when coperfusing cells with subthreshold concentrations of either nucleotide (nt) (100 µM NAADP+3 µM ADPR), we obtained full activation of TRPM2. Here, AMP fully suppressed current activation, confirming that NAADP sensitized TRPM2 gating by ADPR. This prompted us to test whether the NAADP mechanism was related to the one we demonstrated earlier for cADPR. We included the cADPR antagonist 8-Br-cADPR (100 µM), and this too inhibited the response to the co-applied NAADP and ADPR. This clearly suggests that NAADP is an integral part of the response and additionally suggests that its mechanism of action and binding site is the same as that for cADPR.

In light of the synergy between NAADP and ADPR, we asked whether the current activation seen with NAADP alone was entirely due to NAADP or contained an ADPR component that could arise from ADPR mobilization. The full TRPM2 activation by NAADP could be suppressed by both AMP and 8-Br-cADPR. This is consistent with the interpretation that NAADP, like cADPR, has some limited ability to gate TRPM2 directly even when the ADPR component is suppressed by AMP, whereas a direct antagonist like 8-Br-cADPR removes this effect as well.

CONCLUSIONS AND SIGNIFICANCE

Our results indicate that TRPM2, a Ca²⁺-permeable nonselective cation channel natively expressed in lymphocytes, can be gated by ADPR and cADPR. We further conclude that NAADP acts in a very similar manner as cADPR in that it possesses a limited ability to gate TRPM2 directly, but strongly potentiates ADPR-mediated activation of the channel. An important observation in support of a synergy between cADPR and NAADP with ADPR is that intracellular administration of cADPR or NAADP apparently is accompanied by elevated levels of ADPR, which is further increased when using Cs⁺-based intracellular solutions. At this point, we cannot ascribe this ADPR mobilization to a particular mechanism. The simplest explanation, that the nucleotides are metabolized to ADPR, faces the paradox that the best-characterized enzyme that could convert cADPR to ADPR, CD38, is an ectoenzyme and, therefore, not an obvious candidate to mediate this conversion. It is conceivable that other cytosolic enzymes yet to be characterized may be responsible for this phenomenon. Another explanation, that ADPR might be released from intracellular compartments, also cannot be readily explained, since the major store for ADPR is presumed to be mitochondrial and its possible release via cADPR or NAADP has not yet been documented.

An important question relates to the potency and physiological significance of cADPR- and NAADP-mediated activation of TRPM2, which occurs at relatively high concentrations of the second messengers; considerably higher than the nanomolar levels required to activate Ca²⁺ release. First, the nucleotide concentrations needed to activate TRPM2 in our patch-clamp experiments may not necessarily reflect the effective concentrations required to gate TRPM2 in intact cells. The differences we observed with K⁺- and Cs⁺-based pipette solutions attest to the fact that the intracellular environment does affect the sensitivity of TRPM2, and this is inevitably perturbed by whole-cell perfusion. Second, although recent evidence indicates that the receptor agonist cholecystokinin (CCK) can rapidly produce transient increases in NAADP and both CCK and acetylcholine can generate long-lasting increases in intracellular cADPR levels (27), there is no detailed knowledge about the global cytosolic or local subplasmalemmal concentrations that are achieved under specific physiological or pathological conditions. cADPR and NAADP may have a dual mode of action in that low concentrations can trigger Ca²⁺ release and higher concentrations additionally recruit TRPM2. Such a dual function of release activity and Ca²⁺ influx across the plasma membrane is not without precedent, as it has been demonstrated for the vanilloid receptor VR1.

View larger version (26K): [in this window] [in a new window]   Figure 3. Proposed signaling mechanisms for TRPM2 Activation. Release or production of ADPR by mitochondria or the ectoenzyme CD38, respectively, act in synergy with cADPR, NAADP, or H2O2 to activate plasmalemmal TRPM2 ion channels.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5538fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5538fjev1 20/7/962    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Beck, A. Articles by Penner, R. Search for Related Content PubMed PubMed Citation Articles by Beck, A. Articles by Penner, R.