Two-pore channels: going with the flows

In recent years, our understanding of the structure, mechanisms and functions of the endo-lysosomal TPC (two-pore channel) family have grown apace. Gated by the second messengers, NAADP and PI(3,5)P2, TPCs are an integral part of fundamental signal-transduction pathways, but their array and plasticity of cation conductances (Na+, Ca2+, H+) allow them to variously signal electrically, osmotically or chemically. Their relative tissue- and organelle-selective distribution, together with agonist-selective ion permeabilities provides a rich palette from which extracellular stimuli can choose. TPCs are emerging as mediators of immunity, cancer, metabolism, viral infectivity and neurodegeneration as this short review attests.

. Second-messenger synthesis couples plasmalemmal receptors to endo-lysosomal TPCs. A model whereby cell-surface receptors can promote the synthesis of either second messenger, the dinucleotide, NAADP or lysosome-specific lipid, PI(3,5)P 2 . Cations (X + ) exit the acidic vesicle when TPCs are gated by NAADP via small accessory proteins, LSm12 or JPT2, or by PI(3,5)P 2 that binds directly to TPCs. The lysosomal lumen is acidic by virtue of the V-H + -ATPase, which is also the primary drive of the luminally positive membrane potential (ΔΨ). In one model, the H + gradient drives Ca 2+ uptake via an unknown exchanger. an inhibitory one (by locking into a closed state) [23] or a stimulatory one [25]. For the other isoform, TPC2 is activated by luminal Ca 2+ [26]. The reason for the discrepancies is unknown.
Thanks to the atomic structures, we know that PI(3,5)P 2 binds directly to TPC1 and TPC2 ( Figure 3) and how this gates the channel [14,15]; mutagenesis of complementary basic amino acids in the binding pocket abolishes activation by the phospholipid [14,15,36]. For NAADP, TPC stimulation is indirect, with NAADP binding to a smaller, accessory protein(s) (Figure 1). Recent screens have finally identified NAADP-binding proteins that mediate the gating of TPC, namely LSm12 [37] and JPT2 [38] (note: JPT2/HN1L was also reported to activate ryanodine receptors (RyRs) [39]). These proteins were unexpected candidates given that LSm12 is an RNA-binding protein and JPT2 is otherwise mechanistically orphaned (although linked to cancers) [40]. Attesting to its importance, LSm12 deletion is embryonic lethal [37]. Where and how these proteins bind to TPCs (and whether there is any isoform selectivity [40]) will prove a key future direction.
The potential for two molecular messengers to converge upon one channel is unusual, and the physiological consequence of this duality is ill-defined. Whether either (or both) messengers is required for TPC activation requires further work and may also be context-sensitive. For example, in the same macrophage, TPC2 responds Figure 2. Distribution of TPC channels throughout the endo-lysosomal system. TPC1 channels are predominantly found in less acidic, earlier compartments, whereas TPC2 is found in later, more acidic vesicles. Trafficking of cargo through the endo-lysosomal system (e.g. endocytosis, viruses, bacterial toxins) as well as vesicle movement and fusion is also subject to TPC control.
to PI(3,5)P 2 for macropinosome resolution (i.e. shrinkage and resorption) [41], but to NAADP for TPC-dependent phagocytosis [42]. Moreover, inhibition of PI(3,5)P 2 synthesis with vacuolin-1 [43] did not alter NAADP-induced Ca 2+ release [44] implying there is little interaction between the messengers, at least in fibroblasts. The messengers' kinetics, uniqueness, redundancy or potential synergy may shape the signalling palette from which stimuli can choose.  Models depicting the relative permeabilities to Ca 2+ , Na + and H + are conveyed by the size of the coloured plumes. For TPC2, soluble NAADP evokes TPC2 currents with comparable Ca 2+ and Na + conductances, whereas the lipid PI(3,5)P 2 stimulates Na + -selective currents.
When a Na + conductor, TPC1 modulates vesicular ΔΨ and electrical excitability [24]. In an osmotic modality, TPC1/TPC2 co-ordinate macropinosome resolution when their Na + fluxes drive Cl − co-transport, water movement and pinosome shrinkage [41,53]. As Ca 2+ -permeable channels, TPCs have arguably garnered more attention physiologically (see below). Experimentally, it is currently not trivial to distinguish between the Na + and Ca 2+ modalities of TPC signalling in driving biological processes, in part due to our inability to monitor Na + fluxes in situ.
However, the permeability sequence of TPCs is not immutable and depends on the stimulating messenger. Activation of TPC2 via the PI(3,5)P 2 -pathway promotes a predominantly Na + current (P Ca /P Na ∼ 0.08), whereas the NAADP pathway evokes an eight-fold larger Ca 2+ conductance (P Ca /P Na ∼ 0.65) [54] (Figure 4). TPC1 may also exhibit ligand-dependent permeability, albeit more modestly, with PI(3,5)P 2 shifting the P Ca / P Na from 0.98 to 0.42 [22]. Ligand-induced permeability changes are a unique feature of TPCs and thereby resolve early controversies as to the permeant ions. Thus, by the judicious selection of messenger, TPCs may be recruited to signal via Na + (osmolarity, ΔΨ) or Ca 2+ or pH L .
How NAADP elevated the pH L of acidic Ca 2+ stores was unclear [29,55] until the demonstrations that both TPC1 and TPC2 conduct H + , i.e. efflux pathways from vesicles [22,54] (Figure 5). Therefore, NAADP may signal not just by an increase in cytosolic Ca 2+ , but by a coincident alkalinization of endo-lysosomes. Interestingly, pH L changes parallel the Ca 2+ signals in that H + fluxes are stimulated by the NAADP-but not the PI(3,5)P 2 -pathway with TPC2 [54]. In part via effects on vesicular pH, TPC2 influences melanosome pigmentation [47,56] and autophagy [57]. With NAADP as the messenger, it binds to its accessory protein (LSm12 or JPT2, red hexagons) to evoke local Ca 2+ nanodomains that are uniquely sensed by closely associated Ca 2+ -binding proteins ('decoders', brown hexagon)). When PI(3,5)P 2 is the stimulus, Na + -selective currents are evoked which can depolarise the lysosome (ΔΨ) or promote osmotic changes and vesicle shrinkage (by Cl − co-transport and concomitant water loss). NAADP can also promote H + efflux through TPC2 and increase the lysosomal luminal pH ( pH L ).
In summary, different messengers evoke different ionic signals. This may explain, for example, why PI(3,5)P 2 is selected for macropinosome resolution: the lipid favourably stimulates fluxes of Na + (but not H + ) to drive Cl − and water loss [41]; NAADP would have been unfavourable since it evokes smaller Na + fluxes and an increase in pH L that could inhibit the essential Cl − co-transport [53].

Pharmacology
Compounds that modulate TPCs are a growing family, with currently more inhibitors to choose from than activators.
In terms of antagonising the messengers, the only cell-permeant NAADP antagonists that we have are BZ194 [67], the original Ned-19 [68], and its minimally modified analogue, Ned-K [69,70]. We do not yet have any specific inhibitors of the lipid-activation site, although high concentrations of Ned-19 unexpectedly block PI(3,5)P 2 -induced TPC2 currents [58].

Activators
Historically, activation of TPCs in intact-cell populations has been limited to NAADP delivery via liposomes [71,72] or cell-permeant NAADP (NAADP/AM) [73] which is notoriously labile. The recent discovery of stable, cell-permeant agonists that mimic these two TPC activators will open up the field, even if robust Ca 2+ responses require the ectopic expression of TPC2 [54]. Each mimetic targets the TPC2 isoform (TPC2-A1-N [NAADP mimetic] and TPC2-A1-P [PI(3,5)P 2 mimetic]), and are selective for TPC2 over TPC1 and TRPMLs [54]. TPC2-A1-P requires the lipid-binding site on TPC2 [54] (Figure 3)likely a direct interaction with the channelbut the TPC2-A1-N activation mechanism is currently unclear. Does it bind to the NAADP accessory proteins LSm12/JPT2, or does it bind to TPC2 directly and mimic their interactions?
Interestingly, photo-release of another lipid, sphingosine, acutely evoked Ca 2+ signals via TPC1 (but not TPC2) [74]; is this an underexplored new pathway? Surprisingly, tricyclic antidepressants (TCAs) and the motor-neuron-disease medication, riluzole, are TPC agonists [75], but their poly-pharmacology towards other transporters will probably limit their usefulness in intact cells.

Protein regulators
Other signalling inputs may interact with TPCs including protein kinases such as LRKK [80], JNK/p38 [81], mTOR [79] and the small GTPase, Rab7 [82]. Protein kinase A was proposed to modulate TPC2 currents via phosphorylation of Ser666 [46] although, curiously, this residue lies within the lysosomal lumen and not accessible to cytosolic cAMP signals. In some cases, the physiological context for these modifiers is poorly defined.

Polymorphisms
In the global population, TPC2 naturally occurs with a spectrum of different polymorphisms [83] and some impact TPC2 function. The degree of melanin pigmentation is inversely related to TPC2 activity [66] and two gain-of-function (GOF) polymorphisms (in different regions of the TPC2) each promoted blond-hair colour by independent mechanisms [76]. More recently, it was shown that the M484L mutation required an additional 'permissive' L564P polymorphism [83]. GOF mutants certainly produce larger currents in response to PI(3,5)P 2 [54,76,83] or to the TPC2 agonists TPC2-A1-N and TPC2-A1-P [54]. However, it is less certain which signalling modality of TPC2 these polymorphisms produces the phenotype when roles for Ca 2+ [56], Na + (ΔΨ) [47,83] and pH [47,56,83] have all been posited for pigmentation.
How local TPC Ca 2+ signals are otherwise decoded is underexplored. Privileged TPC-coupling to downstream processes implies that Ca 2+ -sensitive decoding proteins are intimately associated with TPCs and sense these high Ca 2+ nanodomains. Several TPC interactomes have been published (reviewed in [93]), but surprisingly few Ca 2+ -binding proteins have been pulled out (e.g. annexins, although interactions have not always been validated). Phagocytosis is uniquely driven by local Ca 2+ from TPCs (but not global Ca 2+ signals) [42], where the Ca 2+ -dependent phosphatase, calcineurin, may be the Ca 2+ decoder [42].

TPCs and health
Our appreciation of the importance of TPCs is expanding, but the following, recent examples incidentally reinforce that the precise molecular details of the circuitry are often lacking and we do not know the messenger, permeant ion or the decoders. Our understanding of the roles of TPCs is still in its infancy.
In the vasculature, the role of TPCs is growing and, in particular, at vasculogenesis. The proliferation of endothelial precursors cells is dependent upon NAADP and TPC1 [72,103], and several studies implicate TPC2 in angiogenesis e.g. [65,104]. During embryogenesis, TPC1 and TPC2 promote different aspects of muscle development, namely myoseptal junction formation [105] and myogenesis, respectively [106]. Moreover, innervation of the muscle likewise relies on the NAADP/TPC2 axis [102].
Metabolically, the nutrient-sensing kinase complex of mTOR inhibits TPC2 which thereby responds to nutrient status [79]. Reciprocally, TPC2-KO enhances mTOR activity [107]. Manipulation of TPC1 expression reveals a potential link to glucose and fat metabolism [108], and the net surface expression of GLUT1 [94] and GLUT4 [108] glucose transporters are under the control of endosomal TPC1, probably by regulating endocytosis. Deletion of TPC2 in mice exacerbates the effects of a high-fat diet by reducing cholesterol/triglyceride clearance [87,109], although this does not translate into weight gain [109], partly due to enhanced insulin sensitivity in the absence of TPC2 [109].
TPCs are abundantly expressed in immune cells and are involved in often complex Ca 2+ circuitry to regulate vesicular trafficking events in an immune context [110]. Extracellular particle clearance and fluid sampling during 'inward' trafficking events like phagocytosis [42] and macropinocytosis [41] in macrophages are mirrored by TPCs controlling 'outward' events like exocytotic secretion of histamine in mast cells [63], of cytolytic factors in cytotoxic T-cells [84] and the surface-presentation of chemokine signalling molecules [111]. Thus, TPCs may be invaluable during anaphylaxis, pathogen clearance by the innate immune system and T-cell clonal expansion.

TPCs and disease
Given their physiological rolesparticularly of membrane and protein trafficking -TPCs are implicated in a wide range of diseases that are a significant health burden [112]. For neurodegenerative conditions like Alzheimer's (AD) and Parkinson's (PD) diseases, endo-lysosomes seem to play a critical role [113]. Accordingly, TPC2, in particular, has been linked to both AD [114] and PD [115,116], perhaps a result of aberrant trafficking, and LRKK2 mutation in the case of PD.
Pathogens deliver toxins and/or enter the host cells to replicate, often gaining access via the endocytic pathway where they traffic through the endo-lysosomal system by co-opting host pathways. Since TPCs regulate vesicular uptake pathways (endocytosis, macropinocytosis, phagocytosis) [41,42,123] and are important for trafficking [86,87,124], their importance in contributing to pathogenicity was likely. Accordingly, inhibiting TPCs reduces infectivity of the Ebola virus [58], and of the Coronaviruses causing MERS [89] and Covid-19 [125,126]. Likewise, reducing the expression of the essential NAADP-binding protein, JPT2, also reduces viral uptake [38]. For HIV-1 replication, the virus subverts TPCs to allow essential Tat protein release [127]. Bacteria require toxins to traffic through the endo-lysosomal system and those for cholera, diphtheria and anthrax rely on TPCs [123,124,128].
An increasing field is that of TPCs in cancer [129] where, remarkably, TPCs impact different aspects. Feeding the tumour requires a blood supply and TPCs help drive angiogenesis [65,104]. Metastatic invasion and migration of the tumour cells themselves is another TPC-dependent process [66,104,130], and finally, tumour proliferation is under TPC2 control [64,66]. Consequently, TPC inhibition reduces tumour mass [64,104], and TPC1 and TPC2 may differentially contribute [131]. It is germane that the NAADP-binding protein, JPT2, is implicated in cancer progression [40].

Conclusion
In this brief overview, we have highlighted the diversity of both the ionic nature of the TPC signals and the breadth of the ( patho)physiological processes in which TPCs play an important role, and this is only set to grow. A common theme is the involvement of TPCs in vesicular trafficking. With still so many unknowns, and the likely intersection with hitherto unsuspected pathways, the field of endo-lysosomal ionic signalling will continue to be a rich source to mine.

Perspectives
• TPCs are endo-lysosomal Ca 2+ -permeable channels that are emerging as important signal transducers across biology and phyla.
• Unusually, their ion conductances depend on the stimulus: they are plastic channels.
• Different conductances confer the ability of TPCs to signal in different modalities (e.g. Ca 2+ , electrical, lysosomal pH).
• New TPC protein regulators have recently emerged.
Competing Interests