ReviewThe endo-lysosomal system as an NAADP-sensitive acidic Ca2+ store: Role for the two-pore channels
Introduction
Changes in the concentration of cytosolic Ca2+ regulate a plethora of cellular events [1]. The importance of this pathway is exemplified by the many diseases that result from mis-regulated Ca2+ signals [2]. Given its pleiotropic actions, it is not surprising that changes in cytosolic Ca2+ concentration are tightly regulated. This is achieved through a rich portfolio of Ca2+ channels, pumps, transporters and buffers which underpin the spatio-temporally complex changes in Ca2+ concentration that typically result upon cell stimulation [3]. Fine tuning of these signals is thought to maintain the specificity of Ca2+-linked stimuli in regulating down-stream targets [1], [3].
Many hormones and neurotransmitters evoke changes in cytosolic Ca2+ concentration through the production of intracellular messengers which, in turn, activate Ca2+-permeable channels located on intracellular stores [1], [3]. By far the best studied of these pathways is that involving inositol trisphosphate (IP3). IP3 is produced by receptor-mediated activation of phospholipase C and mobilizes Ca2+ from endoplasmic reticulum (ER) Ca2+ stores through a well defined family of IP3-sensitive Ca2+ channels [4], [5], [6]. Cyclic ADP-ribose (cADPR) is another Ca2+-mobilizing messenger. It is produced by ADP-ribosyl cyclases and activates ryanodine receptors [7], [8]. IP3 and ryanodine receptors are structurally and functionally related and both reside on ER Ca2+ stores [4], [5], [6], [8]. A key feature of these channels is their biphasic regulation by cytosolic Ca2+ whereby low concentrations stimulate channel activity while higher concentrations inhibit [4], [5], [6], [8]. This self regulatory mechanism is critical for the generation of spatiotemporally complex Ca2+ signals [1].
The most recently discovered Ca2+-mobilizing messenger is nicotinic acid adenine dinucleotide phosphate (NAADP) [9], [10]. The Ca2+-mobilizing properties of NAADP were first recognized by Lee and colleagues using egg homogenates from sea urchin [11]. Much less is known about this pathway compared to IP3 and cADPR pathways. NAADP is produced upon cell stimulation with Ca2+-mobilizing agonists [12], [13], [14] although its mechanism of synthesis is uncertain. Like cADPR, NAADP may be synthesized by ADP-ribosyl cyclases [15], [16]. Perhaps the most surprising feature of NAADP is its ability to target Ca2+-permeable channels on acidic Ca2+ stores that are clearly distinct from the ER [17]. Nevertheless, in intact cells, activation of these channels results in further release of Ca2+ from the ER [18]. NAADP is thus considered a “trigger” whereby it provides local release of Ca2+ that sensitizes neighbouring IP3 and ryanodine receptors to their respective messengers resulting in a larger Ca2+ release event (Fig. 1) [19].
The NAADP pathway is recruited in an agonist-selective manner notably by the same agonists that were thought previously to couple exclusively to IP3 production [20]. These include endothelin-1 [13], cholecystokinin [14], and glutamate [21]. Such differential recruitment of intracellular Ca2+ release channels by different extracellular cues provides a plausible basis for generating heterogeneity in the Ca2+ signal. The resulting Ca2+ signals have been implicated in a variety of cellular events including fertilisation [12], [22], neuronal growth [23] and blood pressure control [24]. In this review, we discuss the evidence that NAADP mediates Ca2+ release from the endo-lysosomal system through activation of a novel family of Ca2+-permeable channels—the two-pore channels (TPCs).
Section snippets
NAADP mobilizes Ca2+ from endo-lysosomal Ca2+ stores
From early studies in sea urchin eggs, it was clear that the Ca2+ stores targeted by NAADP were distinct from the ER [25]. Thus, fractionation of egg homogenates on density gradients resulted in a broad distribution of vesicles that were capable of responding to NAADP [25]. In contrast, IP3- and cADPR-sensitive vesicles strictly co-migrated with markers for the ER [25]. In addition, thapsigargin, which depletes ER Ca2+ stores, effectively abolished Ca2+ release in response to IP3 and cADPR, but
NAADP activates two-pore channels
Considerable progress in defining the NAADP-sensitive Ca2+ store was not, until recently, matched by progress in defining the molecular target of NAADP. In the last year or so, however, three independent groups have converged on the TPCs as likely NAADP targets.
TPCs were cloned in 2000 by Ishibashi et al. from rat [38] and by Furuichi et al. from Arabidopsis [39]. Both proteins display significant sequence similarity (albeit modest) to voltage-gated Ca2+ and Na+ channels [38], [39]. The
Sub-cellular targeting of TPCs
Befitting their role as NAADP-sensitive Ca2+ channels responsible for Ca2+ release from acidic Ca2+ stores, animal TPCs localize to the endo-lysosomal system. In our studies using heterologously expressed TPCs, we found a punctate intracellular distribution for human TPCs [41]. The acidic nature of the labelled vesicles was confirmed by the overlap in distribution of the TPCs with lysotracker red, a fluorescent weak base [45]. Co-expression with markers for endosomes (rhoB) and lysosomes
Structure of TPCs
Based on their sequence similarity to voltage-gated Ca2+/Na+ channels, TPCs are predicted to comprise two homologous domains each consisting of 6 trans-membrane regions with a putative pore-forming domain located between the 5th and 6th membrane-spanning regions [38], [39]. They thus have a unique predicted structure corresponding to approximately half of a voltage-sensitive Ca2+/Na+ channel (Fig. 3A). Consistent with this predicted topology, fluorophores placed at either terminus or after
Electrophysiological properties of TPCs
The electrophysiological characterization of TPCs is at present more advanced in higher plants than animals. Plant TPCs form the ubiquitous SV (slowly activating vacuolar) channels within the vacuolar membrane. SV channels are the most abundant ion channels in the vacuolar membrane and so amenable to patch-clamp recording from isolated vacuoles [40], [62], [63]. They are almost entirely cation-selective with large single-channel conductances (γK ∼ 155pS) and they are Ca2+-permeable, but select
Functional roles of TPCs
NAADP has been shown to regulate a variety of cellular functions that include muscle contraction and differentiation. Here we discuss the role of TPCs in these established NAADP-dependent processes, and also their role in trafficking and pigmentation in which NAADP signalling had not previously been implicated.
Much evidence indicates that NAADP regulates muscle contractility. In pulmonary artery smooth muscle cells, NAADP generates local Ca2+ signals from bafilomycin A1-sensitive Ca2+ stores.
Evolution of TPCs
As mentioned above, structurally speaking, the TPCs correspond to approximately one-half of a voltage sensitive Ca2+/Na+ channel. Since the latter are thought to have evolved from K+ channels [92], TPCs may represent an evolutionary intermediate between the two. Thus the single repeat of the K+ channels may have been duplicated to generate TPCs, which may then have undergone another round of duplication to generate 4-repeat Ca2+/Na+ channels (Fig. 4A).
A distinguishing feature between plant and
Outlook
The molecular basis for Ca2+ release by NAADP has been subject to controversy [99], [100], with competing claims for activation of Ca2+-permeable channels located on the ER, the plasma membrane, and as discussed in this review, acidic Ca2+ stores. Multiple lines of evidence from independent groups are now converging on the TPCs as the target for NAADP on acidic stores of Ca2+. Indeed, the localisation of TPCs to the endo-lysosomal system, within which there is much protein trafficking, might
Acknowledgements
We thank Dev Churamani, Robert Hooper, Chi Li and Desmond Tobin for useful discussion, and the Bogue foundation (University College London) for facilitating staff exchange. Work in SP's laboratory is funded by the BBSRC. Work in CWT's laboratory is funded by the Wellcome Trust. TR is a Drapers’ Research Fellow at Pembroke College, Cambridge.
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2015, Journal of Biological ChemistryCitation Excerpt :In addition, by analogy with the situation in the ER (83), uptake of Ca2+ into acidic stores might be enhanced by glucose-stimulated ATP production, and because luminal Ca2+ sensitizes TPCs to low NAADP concentrations, this could promote Ca2+ release from these stores (84, 85), an effect that would be enhanced by removing competing ER stores. Since the initial reports linking NAADP-evoked Ca2+ release to two-pore channels (29–31), there have been numerous reports of TPCs playing an essential role in mediating NAADP-evoked Ca2+ release from acidic stores (58, 86). However, recent evidence points to a separate NAADP-binding protein that interacts with TPCs to confer NAADP sensitivity (39).
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