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Neutrophil Signalling Group, Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, UK
1Correspondence: Neutrophil Signalling Group, Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK. E-mail: hallettmb{at}cardiff.ac.uk
ABSTRACT
The regulation of non-voltage-operated Ca2+ channels in the plasma membrane remains unclear (1–3). However, there is often a link between the physiological release of Ca2+ from intracellular stores and opening of Ca2+ influx channels on the plasma membrane. This route has been referred to variously as store-operated Ca2+ entry (SOC), capacitative Ca2+ entry, and Ca2+ release-activated channel opening (CRAC), and often underlies the large changes in cytosolic free Ca2+ that accompany many stimuli in a wide variety of cell types (1–3). The linkage between Ca2+ store release and opening of Ca2+ channels on the plasma membrane has remained elusive for a number of years, perhaps in part because different mechanisms exist for this linkage, and are used to differing extents by different cells. We suggest here that one of the mechanisms that may operate in cells of the immune system, but that may be important elsewhere, involves the release of mitochondrial adenosine diphosphate ribose (ADPR) or nicotinamide adenine dinucleotide (NAD+). There is accumulating evidence to support each of the steps necessary for a complete description of this "Ca2+ store release to plasma membrane channel opening" link, but to our knowledge they have not been connected before to make a coherent model.—Ayub, K., Hallett, M. B. The mitochondrial ADPR link between Ca2+ store release and Ca2+ influx channel opening in immune cells.
Key Words: non-voltage-operated Ca2+ • mitochondrial Ca2+ • Ca2+ influx channels
THE HYPOTHESIS
WE PROPOSEthe following model: 1) Ca2+ released from storage sites is "sensed" and a proportion taken up by strategically placed mitochondria; 2) the elevated mitochondrial Ca2+ results in the generation and release of mitochondrial ADPR (or NAD+); 3) elevated ADPR (or NAD+) diffuses to the plasma membrane and acts to open Ca2+-permeable channels that allow Ca2+ influx (Fig. 1
). The evidence for each step is presented.
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MITOCHONDRIA AS A Ca2+ RELEASE SENSOR
It is well known that Ca2+ is taken up by mitochondria as a result of the mitochondria membrane potential through a uniporter (4
, 5)
that has specificity for Ca2+ ions (6)
. An elevation in cytosolic free Ca2+ near a mitochondrion thus results in an elevation in intramitochondrial Ca2+ (7)
. In some cells, some mitochondria are positioned extremely close to the sites of IP3 receptor and Ca2+ release from ER and specifically sense released Ca2+ (8
, 9)
. The proximity of the mitochondria to IP3 receptors (release channels) can be such that mitochondria respond to the release without any detectable changes in bulk cytosolic free Ca2+ (8
, 9)
. As there is evidence in neutrophils that a subset of IP3 receptors exists that is key to triggering Ca2+ influx but inaccessible to inhibition by larger molecular weight inhibitors (10)
, it may be speculated that these IP3 receptors are those at the ER-mitochondria junction and that these play an important role in store-operated Ca2+ influx.
MITOCHONDRIA AS A SOURCE OF ADPR
A major and possibly only source of ADPR within the cell is from NAD+ trapped within the mitochondria (11
, 12)
. ADPR (see structure in Fig. 2
) is generated from NAD+, which is almost exclusively an intramitochondrial molecule, with at least 75% of all cellular NAD+ being within the mitochondria (11)
. NAD+ is normally retained within the mitochondrial matrix because the inner membrane of the mitochondrion is completely impermeant to NAD+ (11
, 12)
. The NAD-ase responsible for the conversion of NAD+ to ADPR, however, is located in the outer part of the mitochondria (13)
(Fig. 2)
. An increase in the inner mitochondria membrane permeability to NAD+ would thereforepermit the production of ADPR, which would be free to diffuse into the cytosol through the permeable outer mitochondrial membrane. Such a change in inner mitochondria membrane permeability can occur as a result of Ca2+ uptake by the mitochondrion. For example, the addition of Ca2+ to isolated mitochondria leads to a marked decline in mitochondrial NAD+ (14)
, an effect ascribed to the opening of the mitochondrial permeability transition pore (MTP). This pore allows release of molecules of up to 1500 kDa (15)
and so would permit the release of NAD+. Cyclosporin A, an inhibitor of the MTP has been shown to completely inhibit Ca2+-mediated hydrolysis of mitochondrial NAD+ and the subsequent generation of ADPR from isolated mitochondria (13)
. Opening of the MTP is usually thought of as a catastrophic event that results from Ca2+ "overload" (15)
, as the experimental conditions required for its opening require "high" Ca2+. However, some mitochondria within the cell may be subjected to a much higher Ca2+ microenvironment than seen in the bulk cytosolic Ca2+ (8
, 9
, 16)
. Indeed, there is evidence that these mitochondria are ideally placed to sense released of Ca2+ in preference to bulk Ca2+ (8
, 9)
and that the concentration of Ca2+ within the "mitochondrial-ER (m/ER) synapse" may be very high (16
, 17)
. The buffering effect of experimental Ca2+ chelators on the high Ca2+ within the m/ER synapse thus may be minimal and explain why store-operated Ca2+ influx persists in "BAPTA-loaded" cells (3)
.
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NAD/ADPR-SENSITIVE Ca2+ INFLUX CHANNELS
TRP or TRP-like channels have been speculated to be part of the capacitative Ca2+ influx route (18)
. Immunocytes (19)
and myeloid cells such as U937 cells (20)
, neutrophils, and HL60 s cells (21
, 22)
express such a channel, TRPC 7, also known as LTRPC2. Like other TRP channels, it has relatively poor selectivity for Ca2+ and can be permeated by a number of ions. Ca2+ influx into neutrophils and lymphocytes lacks Ca2+ selectivity, being associated with the influx of other ions including Mn2+ and Ba2+ (but not Ni2+). The LTRPC2 channel is opened in response to ADPR (and NAD+), which act through a NUDIX-like motif on the channel (20)
. When expressed in cells not normally expressing these channels, increased conductance in response to ADPR is marked (22)
. In immune cells that naturally express this channel, lymphoid cells (such as T lymphocytes) and myeloid cells (such as neutrophils), elevation of cytosolic ADPR increases Ca2+ influx and elevates cytosolic free Ca2+(21
, 22)
. There is no overlap in the activity of ADPR with the Ca2+-releasing molecule cyclic ADP-ribose, the latter releasing Ca2+ from membranous stores within some cells but having no effect on this plasma membrane channel (21
, 22)
. In contrast, ADPR (and NAD+) has no effect on releasing Ca2+ from membranous stores, being specific for opening LTRPC2 channels at the plasma membrane. Although the existence of the ADPR (NAD+) -activated channel in cells of the immune system is becoming increasingly recognized, its physiological function will remain obscure without a mechanism to regulate its activators. It seems unlikely that the concentration of cytosolic NAD+ would have a large dynamic range, whereas the fold increase in cytosolic ADPR released from mitochondria could be dramatic and act as a Ca2+ channel-opening signal.
SUPPORT FOR THE ROLE OF MITOCHONDRIA AS A LINK IN SOC OPENING
Hoth’s (23)
and Parekh’s (24)
laboratories have provided evidence that links the membrane potential of mitochondria to a role in signaling Ca2+ influx.
In both lymphoid and myeloid cell lines, "energized" mitochondria play an important role in the opening of the SOCs. Several mechanisms have been suggested. For example, mitochondrial Ca2+ uptake may lower local cytosolic Ca2+ near the Ca2+ influx channel and thus prevent Ca2+ inactivation of the Ca2+ influx channel (25)
. Alternatively, mitochondrial Ca2+ uptake may assist in emptying storage sites of their Ca2+ by acting as a Ca2+ "sink" to lower store Ca2+ below a critical level that, in some way, leads to activation of the SOCs (25)
. However, these effects alone are unable to account for the role of mitochondria in Ca2+ influx, as experimentally collapsing the mitochondria membrane potential at any time after release of store Ca2+ also inhibits Ca2+ influx (24
, 26)
. It is striking that this effect is not mimicked by inhibition of mitochondrial ATP synthesis (24)
and no evidence for a role for ATP itself has been found (27)
. Together, these data point strongly to the maintenance of mitochondria potential as necessary to signal Ca2+ influx and are consistent with a role for the generation of a mitochondrial factor for continued Ca2+ influx. Thapsigargin, which inhibits SERCa pumps and permits Ca2+ to leak through ER translocons (28)
(rather than the IP3 receptor channel), also triggers Ca2+ influx. However, it is possible that because of the dependency of IP3 receptors on Ca2+, the local leak of Ca2+ caused by thapsigargin permits existing low levels of IP3 to become effective. In basophils, this Ca2+ influx is stimulated only after many minutes of thapsigargin treatment. Another experimental strategy for depletion of cellular Ca2+ (and presumably stored Ca2+) by prolonged incubation with Ca2+ chelators can result in Ca2+ channel opening in myeloid and lymphoid cells (29
30
31)
. Neither experimental procedure necessarily mimics a physiological routes, and they are unlikely to use the mitochondrial ADPR mechanism proposed here. Indeed, the ICRAC channels that open in thapsigargin-treated basophils are unlikely to be LTPRC2 channels (3)
, although their opening is dependent on energized mitochondria (24)
. A role for the MTP can be suggested from indirect evidence of the effects of a blocker of the mitochondrial transition pore, cyclosporin A (which inhibits the release of mitochondrial ADPR), which has effects consistent with an effective blocker of SOC channel opening in T cells and myeloid cells (32
33
34)
. Opening of MTP may explain the mitochondrial depolarization observed during Ca2+ release (35
, 36)
.
SUPPORT FOR THE ROLE OF A DIFFUSIBLE FACTOR IN IMMUNE CELLS
The key feature of this hypothesis is that the opening of ADPR-sensitive Ca2+ channels is linked to Ca2+ store release by a diffusible factor, ADPR. In lymphoid cells (e.g., Jurkats and T cells) and in myeloid cells (e.g., neutrophils and HL60 s), there is evidence of a diffusible link between Ca2+ store release and Ca2+ influx (37–39). It is possible to extract a factor from Jurkat cells (37)
or neutrophils (39)
that has some of the properties required for this diffusible link. Although this extract has other properties (39
40
41)
, the molecular mass of the fraction that has Ca2+ channel-opening properties has been estimated at between 500 and 800 Da (37
, 40
, 41)
. Again, this is consistent with an identity of either ADPR, whose molecular mass is 559.3 Da, or NAD+ (mol wt=663.4) as the diffusible link for store operated Ca2+ influx.
CONCLUSIONS
We therefore propose that mitochondrial ADPR generation is the link that connects Ca2+ release to opening of Ca2+ influx channels and accounts for capacitative Ca2+ entry in immune cells and possibly in other cells. Of course, even in immune cells it is likely that other pathways linking Ca2+ store release also exist; nevertheless, the components for all the steps of this pathway exist and probably operate in immune cells.
Received for publication March 18, 2004. Accepted for publication May 10, 2004.
REFERENCES
This article has been cited by other articles:
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  A. Gasser, G. Glassmeier, R. Fliegert, M. F. Langhorst, S. Meinke, D. Hein, S. Kruger, K. Weber, I. Heiner, N. Oppenheimer, et al. Activation of T Cell Calcium Influx by the Second Messenger ADP-ribose J. Biol. Chem., February 3, 2006; 281(5): 2489 - 2496. [Abstract] [Full Text] [PDF]
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