REVIEW CELL BIOLOGY

Function and dysfunction of two-pore channels Sandip Patel Two-pore channels (TPCs) are evolutionarily important members of the voltage-gated ion channel superfamily. TPCs localize to acidic Ca2+ stores within the endolysosomal system. Most evidence indicate that TPCs mediate Ca2+ signals through the Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) to control a range of Ca2+-dependent events. Recent studies clarify the mechanism of TPC activation and identify roles for TPCs in disease, highlighting the regulation of endolysosomal membrane traffic by local Ca2+ fluxes. Chemical targeting of TPCs to maintain endolysosomal “well-being” may be beneficial in disorders as diverse as Parkinson’s disease, fatty liver disease, and Ebola virus infection. Introduction

TPCs as NAADP Targets

TPCs are unusual in localizing to acidic organelles, such as endosomes and lysosomes in animal cells (12–14) and vacuoles in plants (15). This is achieved through dileucine targeting motifs for select isoforms (16, 17). Many acidic organelles are rich in Ca2+. These so-called acidic Ca2+ stores mediate signaling throughout kingdoms (18–20). In animal cells, the Ca2+mobilizing messenger nicotinic acid adenine dinucleotide phosphate

Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK. E-mail: [email protected]

Controversy

Despite confirmation of a role for TPCs in NAADP action from several independent laboratories (46–51), some reports have provided evidence to the contrary (52–55). Using a lysosomal patch-clamp method, Wang et al. identified a pronounced Na+-selective current activated by the endolysosomal phosphoinositide PI(3,5)P2 (phosphatidylinositol-3,5-bisphosphate) (52).

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Two-pore channels (TPCs) belong to the superfamily of voltage-gated ion channels (1). TPCs were originally identified in animals (2) and plants (3) on the basis of sequence identity with voltage-gated Ca2+ (Cav) and Na+ (Nav) channels. Cav and Nav channels are targeted clinically by antihypertensives and local anesthetics, respectively (4). Voltage-gated ion channels have a modular structure comprising combinations of pore-forming regions with two transmembrane (TM) helices and voltage sensors with four TM helices, which are often concatenated to form six TM domains (1). TPCs hold a key position in the evolution of voltage-gated ion channels because of their likely dimeric nature (5, 6) and the duplicated domain (2 × 6 TM) architecture of each subunit (7) (Fig. 1A). This structural organization places TPCs between tetrameric one-domain (6 TM) channels, such as CatSper channels and voltage-gated K+ channels (Kv), and monomeric four-domain (4 × 6 TM) channels, such as Cav and Nav channels. The latter are thought to have evolved through sequential rounds of intragenic duplication from an ancestral one-domain precursor (8). Phylogenetic analysis using individual domains of TPCs and those of Cav and Nav channels confirms that fourdomain channels likely arose from an intragenic duplication event and that individual domains of the TPCs are more closely related to respective individual domains within Cav and Nav channels than to each other (Fig. 1B) (9). Such kinship likely underlies the “loose” pharmacology of TPCs whereby drugs that bind either Cav or Nav channels inhibit TPCs, possibly through a common site (9). The presence of Cav-like selectivity filters in a previously unknown clade of TPCs in unicellular organisms suggests that the structural determinants underlying both pharmacological block and ion selection may have been in place in an ancient two-domain precursor, that is, before the postulated intragenic duplication event that gave rise to extant four-domain channels (9). Lineage-specific loss of TPCs, even within closely related species (humans and mice have two TPC genes, whereas other primates and rodents have three) (10, 11), points to ancestral, isoformspecific physiological functions.

(NAADP) (21) plays a key role in controlling Ca2+ signaling by such stores, which are often mobilized in conjunction with the better-characterized Ca2+ stores of the endoplasmic reticulum (ER) (22–24). The bulk, but not all, of the evidence suggest that NAADP mediates Ca2+ release from acidic organelles through an intracellular Ca2+-permeable channel that is distinct from inositol trisphosphate (IP3) receptors and ryanodine receptors, both of which are localized to the ER (24). This is perhaps best exemplified in sea urchin egg homogenates, where the Ca2+-mobilizing properties of NAADP were discovered (25–27). NAADP-evoked Ca2+ signals in intact cells are thought to be amplified by IP3 and ryanodine receptors (28), possibly at regions of close apposition (membrane contact sites) between acidic organelles and the ER (29, 30). Progress in the field was hampered by the lack of a molecular correlate for the NAADP-sensitive Ca2+-permeable channel. Although there is evidence that NAADP activates ryanodine receptors and select transient receptor potential channels in some cells or preparations (31, 32), in 2009, three independent groups converged on TPCs as the likely target for NAADP (12–14) (Fig. 2A). Befitting their role as the long-sought target channels for NAADP, overexpression of TPCs potentiated NAADP-evoked Ca 2+ signals (10, 12–14, 16, 33–36). Conversely, gene knockout, small interfering RNA (siRNA) silencing, and dominant-negative TPC mutants abrogated NAADP-evoked signals, functionally associated Ca2+-dependent events, or both (12, 13, 35, 37). TPC2 has also been proposed to regulate store-operated Ca2+ entry (38). Electrophysiological analyses of TPCs have been reported using enlarged lysosomes combined with a planar patchclamp approach (39), by inserting the channels into planar lipid bilayers (40, 41), or with patch-clamp studies of channels redirected to the plasma membrane (16, 42). Such studies confirmed NAADP-activated channel activity, Ca2+ (or Ca2+ surrogate) permeability, and a requirement for conserved residues within the predicted pore region. Luminal Ca2+ activates TPC1 (41) and TPC2 (40). Luminal H+ also activates both TPC isoforms (39, 41), although regulation of TPCs by pH is complex (40). Indeed, a bilayer study suggests that TPC1 exhibits substantial permeability to H+ (43), potentially accounting for NAADP-mediated alkalization of acidic organelles (44). N-glycosylation of residues close to the second pore domain inhibits TPC1 activity (7). Conversely, the trafficking protein Rab7 interacts with the N-terminal portion of TPC2 and enhances TPC2 activity (45).

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Fig. 2. Models for TPC activation and permeability. (A) (Left) Original model based on evidence that TPCs are NAADP-gated Ca2+-permeable channels. (B) Alternative model based on evidence that TPCs are PI(3,5)P2-gated Na+ channels. (C) “Hybrid” model suggesting that TPCs are Na+, Ca2+ (and H+)– permeable channels co-regulated by PI(3,5)P2 and NAADP (through associated NAADP-binding proteins) and blocked by Cav and Nav modifiers.

This current appeared independent of TRPML1 (mucolipin 1), another lysosomal ion channel activated by PI(3,5)P2 (56). Overexpression of TPCs recapitulated the PI(3,5)P2-induced current, and this current was abrogated in double-knockout mice lacking TPC1 and TPC2 (52). Somewhat surprisingly, the authors found no evidence for regulation of TPCs by NAADP and NAADP-mediated Ca2+ signals appeared unaffected in pancreatic b cells from their transgenic mouse (52). The authors concluded that TPCs are not NAADP targets nor Ca2+-permeable but rather are PI(3,5)P2-activated Na+ channels (Fig. 2B). Another study suggested that TPCs associated with the kinase mTOR (mammalian target of rapamycin) and that ATP (adenosine triphosphate)–mediated inhibition of Na+ currents through TPC1 and TPC2 involved this mTOR-TPC complex (53). TPC1 and TPC3 were reported to

four subunits to form a functional tetrameric channel, TPCs form a pseudotetramer by association of two subunits, and Cav and Nav channels form a pseudotetramer with a single subunit. Similarity in domains is indicated by the two colors. (B) Phylogenetic relationships between individual channel domains of TPCs, Cav and Nav. Adapted from (9).

be “classically” voltage-activated and to regulate electrical excitability of endolysosomes (54) and possibly the plasma membrane (55), respectively. TPC3 was insensitive to both PI(3,5)P2 and NAADP (55), similar to plant TPCs (57). Thus, in these studies, the properties of TPCs appeared inconsistent with a role in NAADP-mediated Ca2+ signaling. Recent work, prompted by the above studies, has gone a long way to resolving the permeability and ligand activation of TPCs. Na+ permeability was not assessed in the initial biophysical characterization of TPCs (16, 39–42). Thus, Jha et al. (58) performed similar lysosomal patch-clamp experiments as described previously (52–55) and confirmed that TPC2 is activated by PI(3,5)P2, is Na+-permeable, and is regulated by ATP, at least in part, through mTOR. However, TPC2 was also activated by NAADP, even though current amplitudes were modest in relation to those induced by PI(3,5)P2 (58). Grimm et al. also confirmed activation of TPC2-dependent Ca2+ currents by PI(3,5)P2 (59). Critically, endogenous TPC2-like Ca2+ currents were recorded in response to PI(3,5)P2 and NAADP, and these currents were substantially reduced in embryonic fibroblasts derived from a TPC2-knockout mouse (59). Similar results were obtained in embryonic fibroblasts derived from an independent TPC2-knockout strain (12, 60). Calculation of an Na+/ Ca2+ permeability ratio of close to unity for endogenous TPC2 indicated that TPC2, at least, is not markedly Na+-selective (59, 60). Consistent with this notion is the presence of conserved asparagine residues in the putative selectivity filter of TPCs similar to NMDA (N-methyl-D-aspartate) receptors of the glutamate receptor family, which are Ca2+- and Na+-permeable (9). Rahman et al. applied a pharmacological approach to relate NAADP action to TPCs (9). They identified both Nav and Cav modifiers (including the Nav opener veratridine) as blockers of endogenous NAADP-evoked Ca2+ signals in the “gold standard” sea urchin egg homogenate and correlated such block to predicted interactions with the TPC pore on the basis of molecular docking analyses. NAADP-evoked Ca2+ signals in cells expressing recombinant TPC1 were also inhibited by these blockers (9). Finally, Ruas et al. systematically assessed the contributions of TPCs to NAADP-evoked Ca2+

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Fig. 1. TPCs as evolutionary intermediates. (A) Predicted structure of individual (cylindrical) and assembled (circular) TPC subunits in relation to oneand four-domain channels. The gray curved arrow represents evolution from channels with one domain through TPCs with two domains to Cav and Nav channels with four domains (D1 to DIV). CatSper and Kv channels require

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NAADP regulates various physiological events (24, 68), including cellular differentiation (69), muscle contraction (70), and endothelial cell activation (71). In accord, studies have demonstrated functional requirements for TPCs in these very same processes (37, 48, 72–74). Additional roles for TPCs have been identified in membrane trafficking (33, 45, 63), autophagy (46, 49, 75), nutrient sensing (53), exocytosis (35, 76), angiogenesis (77), fertilization and embryogenesis (78), and cytokinesis (79). Importantly, recent work shows that TPCs are also involved in disease, including Parkinson’s disease (80), fatty liver disease (59), and Ebola infection (81). Although these diseases have rather distinct pathologies, there is a mechanistic convergence on trafficking events within the endolysosomal system. Parkinson’s disease is a common incurable neurodegenerative disorder that is characterized by the loss of dopaminergic neurons in the midbrain.

Lysosomal dysfunction may contribute to disease pathology (82). Hockey et al. examined lysosome morphology in fibroblasts from patients with the common G2019S mutation in LRRK2, which causes an autosomal dominant form of the disease (80). The function of the encoded protein is not entirely clear, but much evidence point to an endolysosomal locus of action (83). Indeed, Gómez-Suaga et al. provided evidence that overexpression of LRRK2 may disrupt autophagy, a process that involves lysosomal fusion with autophagosomes, through a mechanism involving NAADP and TPC2 (84). In LRRK2G2019S–Parkinson’s disease patient fibroblasts, the morphology of the endolysosomal system was markedly disrupted and characterized by clumped and swollen lysosomes (80), which could reflect a trafficking defect within the endolysosomal system. Defects in LRRK2G2019S fibroblasts were reversed by silencing TPC2 but not TPC1 (80). Furthermore, reversal was also achieved by chemically antagonizing NAADP action or inhibiting PI(3,5)P2 synthesis (80). Such findings support the co-regulation model of TPC activation (Fig. 2C). Defects were also reversed by antagonizing Rab7 (80), which interacts with TPCs and mediates endolysosomal morphology defects upon TPC2 overexpression (45). TPCs, therefore, represent a convergence point in the control of membrane trafficking through the endolysosomal system by integrating established protein (Rab-7) and lipid [PI(3,5)P2] cues with ion channel activity (85). Such interactions are of potential therapeutic relevance to Parkinson’s disease, although it is not yet known whether TPCs deregulate trafficking in neurons. Grimm et al. analyzed TPC2-knockout mice fed a cholesterol-rich (“Western-style”) diet and noted several features consistent with nonalcoholic fatty liver disease (59). These included discoloration of the liver and an increase in liver weight and cholesterol. Livers from TPC2-knockout mice also accumulated lipid droplets and showed signs of fibrosis. Additionally, circulating amounts of low-density lipoprotein (LDL) were increased. No differences were observed between wild-type and TPC2knockout mice fed a standard diet. Mechanistically, the authors provided evidence that trafficking of cargo through the endolysosomal system was impaired in the TPC2-knockout mice (59). Thus, both LDL and epidermal growth factor (EGF) accumulated in late endosomes or lysosomes in TPC2deficient mouse embryonic fibroblasts. It will be of major interest to relate these findings to human fatty liver disease. The Ebola virus is a filovirus that causes hemorrhagic fever and is associated with a high mortality rate. It enters cells through macropinocytosis (a form of endocytosis), traffics through the endosomal system, and is thought to be released into the cytoplasm through a mechanism involving the late endosome and lysosome protein Niemann-Pick C1 (NPC1) (86). Sakurai et al. (81) found that cellular infection was inhibited by several Cav blockers, including tetrandrine, which was the most potent of the drugs tested. Cav channels have previously been implicated in virus entry (87), but they are perhaps unlikely the drug targets in these nonexcitable cell models. In support of this, gabapentin proved ineffective in preventing infection, although it should be noted that gabapentin does not target the CaV pore-forming a subunit but rather the associated a2d subunit (88). Cellular infection was reduced by siRNA silencing or knockout of TPC1 or TPC2, overexpression of dominant-negative TPC2, or the NAADP antagonist Ned-19 (81). These data suggest that the beneficial effects of Cav antagonists are likely mediated by blocking NAADPmediated activation of TPCs. Thus, like in fibroblasts from the Parkinson’s disease patients (80), the combined use of molecular and chemical tools has proved most informative. Tetrandrine, which was shown to inhibit NAADPevoked Ca2+ release, TPC-dependent Ca2+ currents, and growth factor trafficking in isolated cells, may block infection by acting at a late stage of virus transit within the endolysosomal system, thereby accounting for virus accumulation in TPC2- and NPC1-positive compartments (81). Tetrandrine proved beneficial in a mouse model of Ebola infection. One prediction

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signals in embryonic fibroblasts derived from mice lacking TPC1, TPC2, or both (60). NAADP-induced Ca2+ signals were significantly reduced upon deletion of either isoform and were essentially eliminated upon double knockout, indicating that both TPC1 and TPC2 are NAADP targets. In these cells, the inhibitory effects of TPC deletion were rescued by reexpression of TPCs but not of TRPML1, which had been proposed as an NAADP target (61). TPC2 point mutants lacking channel activity or with altered Ca2+ permeability (39) also proved ineffective in rescue experiments (60). Together, these data reaffirm the role of TPCs in NAADP action. Possibilities to explain discrepancies in the literature include the proposal that NAADP-mediated activation may be abrogated by introducing fluorophores to the N termini of TPCs. This has been demonstrated for TPC1 (62) and might explain lack of NAADP regulation in the experiments of Zong et al. (14) and Wang et al. (52). Additional experimental considerations relate to Mg2+, which has been reported by Jha et al. to inhibit currents through TPC2 (58) and which may have masked smaller NAADP-induced currents in previous lysosomal patch-clamp studies (52). Persistence of NAADP-induced Ca2+ signals in the double-knockout mice described by Wang and colleagues (52) has been proposed to result from contaminants in the cell-permeable NAADP analog used (48) or incomplete TPC knockout (63). Indeed, N-terminally truncated constructs of TPC1 and TPC2, corresponding to those that could potentially be expressed in the transgenic mice that retained NAADP sensitivity (52), were shown to rescue NAADPevoked Ca2+ signals in an independent TPC double-knockout mouse where NAADP responses were lost (60). Other studies, however, have suggested that the N termini of TPCs are critical for trafficking of TPCs to the endolysosomal system (16, 62). Moreover, although responses to NAADP were not affected upon double knockout of TPCs in the studies of Wang et al., those to PI(3,5)P2 were reduced in a manner consistent with gene loss (52). Alternatively, if truncated TPCs are expressed then this might indicate a requirement for the N-termini of TPCs in PI(3,5)P2 action. Certainly, documentation of TPC transcripts and protein abundance in the various animals used in the field, including the “knockouts,” would be a welcome addition to the literature (64). Finally, insensitivity of TPCs to NAADP might reflect loss of crucial accessory factors. In this context, photo-affinity labeling studies suggest that NAADP does not bind directly to TPCs but rather to small molecular weight binding proteins that associate with TPCs (65–67). In summary, although the bulk of evidence suggest that TPCs are indeed NAADP targets, their mode of activation and permeability is more complex than initially envisaged and is perhaps exquisitely sensitive to experimental configuration. In light of recent findings, I suggest that TPCs are likely co-regulated by NAADP and PI(3,5)P2, permeable to Na+ and Ca2+ (and possibly other cations), and blocked by drugs that bind Nav or Cav channels (Fig. 2C).

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Fig. 3. TPCs and endolysosomal membrane traffic. Schematic highlighting potential loci where local Ca2+ flux through TPCs within the endolysosomal system may modulate fusion or fission events (top) or serve as a trigger for generating global Ca2+ signals in conjunction with Ca2+ release from the ER (bottom).

is that TPC-knockout mice are resistant to infection. This study (81) further points to TPCs as potential therapeutic targets. TPCs, Local Ca2+ Signalling, and Endolysosomal Membrane Traffic

It is currently unclear exactly how TPCs regulate membrane traffic to account for the phenotypes described above. One possibility is that TPCs mediate local Ca2+ fluxes to regulate vesicular fusion or fission events or both (Fig. 3). Indeed, the role of local Ca2+ signals in regulating such events has been appreciated for some time but ascribed mainly to the action of TRPMLs (89, 90). Furthermore, local Ca2+ signals have long been hypothesized to underlie global Ca2+ signals triggered by NAADP during signaling (28). In Parkinson’s disease patient fibroblasts, lysosomal structures are enlarged, an effect that is reversed by inhibiting TPC2 and is associated with exaggerated NAADP-evoked Ca2+ signals (80). These findings point to a gain of function in the NAADP-TPC pathway that may promote homo- or heterotypic fusion of late endosomes and lysosomes in the disease. Defects in patient fibroblasts (and also oocytes overexpressing TPC2) were reversed by the fast Ca2+ chelator BAPTA but not by the slower chelator EGTA (45, 80), pointing to aberrant local Ca2+ signaling through TPC2. Moreover, electron microscopy of patient fibroblasts identified hourglass lysosomal structures not inconsistent with a fusion defect (80). Conversely, TPC2 loss of function may inhibit fusion events and thus prevent the transit of cargos, such as cholesterol, growth factors, and viruses, in TPC2-deficient cells or in cells exposed to Cav blockers (59, 81). Indeed, these defects can be mimicked by BAPTA but not EGTA, again implicating local Ca2+ fluxes in the control of membrane traffic. Proteomic analyses of

Regardless of their exact mechanism of action, TPCs are beginning to be linked to pathology, suggesting that pharmacological targeting of these channels may be clinically beneficial. Presently, there are no selective TPC blockers. However, clinically approved blockers of Cav or Nav channels show efficacy at TPCs (9). Indeed, Cav channel blockers are being evaluated for the treatment of Parkinson’s disease (92). Might “off-target” effects of Cav channel blockers on TPCs prove beneficial with respect to trafficking defects in this disease or others? Ideally, TPC2-selective blockers would be most useful in this context, notwithstanding the potential formation of heteromeric TPC complexes (6). Indeed, TPC isoforms display much less sequence identity even within the most conserved pore region than do Cav or Nav isoforms for which selective blockers have been developed. Such divergence within TPCs could substantially aid drug development. Nevertheless, “pan” inhibitors could be important for combating Ebola infection given that TPC1 or TPC2 silencing prevents virus infection (81). If loss of TPC function precipitates liver dysfunction (59), then it follows that TPC activators might prove beneficial in this disorder just as synthetic activators of TRPMLs can correct trafficking defects associated with hypomorphic function of TRPML1 in the lysosomal storage disorder mucolipidosis IV (93). Potential links between TPCs and Alzheimer’s disease (94), diabetes (95), and obesity (96) further point to TPCs as potential therapeutic targets. REFERENCES AND NOTES 1. F. H. Yu, W. A. Catterall, The VGL-chanome: A protein superfamily specialized for electrical signaling and ionic homeostasis. Sci. STKE 2004, re15 (2004). 2. K. Ishibashi, M. Suzuki, M. Imai, Molecular cloning of a novel form (two-repeat) protein related to voltage-gated sodium and calcium channels. Biochem. Biophys. Res. Commun. 270, 370–376 (2000). 3. T. Furuichi, K. W. Cunningham, S. Muto, A putative two pore channel AtTPC1 mediates Ca2+ flux in Arabidopsis leaf cells. Plant Cell Physiol. 42, 900–905 (2001). 4. F. H. Yu, V. Yarov-Yarovoy, G. A. Gutman, W. A. Catterall, Overview of molecular relationships in the voltage-gated ion channel superfamily. Pharmacol. Rev. 57, 387–395 (2005). 5. D. Churamani, R. Hooper, E. Brailoiu, S. Patel, Domain assembly of NAADP-gated two-pore channels. Biochem. J. 441, 317–323 (2012). 6. K. Rietdorf, T. M. Funnell, M. Ruas, J. Heinemann, J. Parrington, A. Galione, Twopore channels form homo- and heterodimers. J. Biol. Chem. 286, 37058–37062 (2011). 7. R. Hooper, D. Churamani, E. Brailoiu, C. W. Taylor, S. Patel, Membrane topology of NAADP-sensitive two-pore channels and their regulation by N-linked glycosylation. J. Biol. Chem. 286, 9141–9149 (2011). 8. M. Strong, K. G. Chandy, G. A. Gutman, Molecular evolution of voltage-sensitive ion channel genes: On the origins of electrical excitability. Mol. Biol. Evol. 10, 221–242 (1993).

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Cytosolic Ca2+ signal

TPCs converged on components of the fusion apparatus as TPC interactors (45, 59). Both studies identified syntaxins (45, 59), which are SNARE proteins, suggesting an intimate association between a “point source” of Ca2+ (TPCs) and downstream effectors (syntaxin and other as yet unidentified fusogenic proteins). This is similar to the arrangement of Cav channels at the plasma membrane and the exocytotic machinery of synaptic vesicles. Local Ca2+ signaling events through TPCs might also be relevant to other trafficking events, such as retrograde trafficking between endosomes and the Golgi (33), secretion of lysosome-related organelles (35, 76), and, by extension, perhaps even secretion by fusion of conventional lysosomes and endosomes at the plasma membrane (Fig. 3). Endosome-to-Golgi trafficking is a route that is used by cholera toxin and thus also of potential disease relevance. TPCs might also localize to membrane contact sites between endolysosomes and the ER, potentially extending the roles of local Ca2+ fluxes to regulation of nonvesicular trafficking events (91) (Fig. 3). The functional relevance of local Ca2+ signaling through NAADP-mediated activation of TPCs in endolysosomal membrane trafficking is emerging.

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Function and dysfunction of two-pore channels Sandip Patel (July 7, 2015) Science Signaling 8 (384), re7. [doi: 10.1126/scisignal.aab3314]

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