P2X4 is an ATP-gated cation channel that is widely expressed in most tissues in the body and at especially high levels within immune, endothelial, and epithelial cells. This channel plays a role in the secretion of inflammatory mediators, including brain-derived neurotrophic factor and prostaglandin E2, and as a regulator of cardiac contractility and vascular remodeling (Stokes et al., 2017; Suurväli et al., 2017). Within the P2X family, P2X4 has a unique subcellular distribution that is predominantly intracellular, within endolysosomal compartments (Bobanovic et al., 2002; Qureshi et al., 2007). This unusual distribution has sparked a debate about whether it might function at endolysosomal membranes in addition to its role at the plasma membrane. Patch-clamp recordings of ATP-evoked currents from enlarged vacuolar lysosomes have supported this view and revealed that lysosomal P2X4 receptors are under the dual regulation of intraluminal ATP and pH (Huang et al., 2014). The evidence to date suggests that P2X4 is one of several highly Ca2+-permeable lysosomal channels that control lysosomal Ca2+ fluxes and lysosome membrane trafficking events (Cao et al., 2015). Much of this evidence is based, however, on pharmacological manipulation of lysosome pH. In this issue of the Journal of General Physiology, Fois et al. provide a clearer description of the physiological role of lysosomal P2X4 receptors during the secretion of surfactant from alveolar type II (ATII) epithelial cells.

ATII cells are responsible for the secretion of pulmonary surfactant into the lumen of the alveoli. Surfactant is stored within large secretory compartments known as lamellar bodies (LBs), which are considered to be lysosome-related organelles because of their acidic lumen and the presence of many proteins that are associated with conventional lysosomes, including cathepsin D, Rab 11, Lamp-1, and the vacuolar H+-ATPase (V-ATPase; Ridsdale et al., 2011). LBs are not unique to ATII cells; they are also found in other epithelial cells with specialized secretory functions, including keratinocytes. Previous work from the group of Manfred Frick showed that P2X4 is localized to the LBs in ATII cells and plays a critical role in the secretion and activation of surfactant (Miklavc et al., 2011; Thompson et al., 2013). The first step in secretion is the fusion of the LB with the plasma membrane, followed by the opening of a fusion pore. Although this is not sufficient to release surfactant, which is very insoluble and stored in a densely packed membranous structure, it does trigger P2X4 receptors within the LB membrane to activate and generate a highly localized, cytosolic Ca2+ signal in the immediate vicinity of the fused LB (fusion-activated Ca2+ entry [FACE]; Miklavc et al., 2011). FACE drives expansion of the fusion pore and facilitates surfactant release via a mechanism dependent on actin coat contraction and vesicle compression (Miklavc et al., 2012). The P2X4-mediated current also promotes fluid resorption from lung alveoli, which aids the insertion of surfactant into the air–liquid interphase (Thompson et al., 2013).

In their latest paper, Fois et al. (2018) advance this story by demonstrating that the LB itself is the source of ATP that is required to activate P2X4 receptors within the LB membrane once the fusion pore has opened. A key aspect of this study is the ability to correlate the initial fusion event with a jump in extracellular ATP with high temporal and spatial resolution. Two different approaches were used to obtain precise measures of ATP: the first, a genetically encoded ATP sensor attached to a glycosyl phosphatidyl inositol (GPI) anchor (ATeam3.10-GL-GPI); and the second, a microelectrochemical ATP sensor. Both were combined with live imaging experiments to simultaneously record fusion events. What, then, is the trigger for timing the activation of FACE to the initial opening of the fusion pore, given that millimolar ATP is present within the LB? The answer is neutralization of intraluminal pH, which occurs upon opening of the fusion pore. LBs have an acidic pH of ∼5.5, and P2X4 is strongly inhibited under these conditions by virtue of a histidine at position 286 within its external loop, which is not conserved in other members of this family. The dual regulation of P2X4 by pH and ATP prevents the premature activation and desensitization of the receptor, which would otherwise occur in the intact LB.

These latest findings from the Frick group are consistent with several previous reports demonstrating that lysosomes contain high levels of ATP. Early evidence was obtained in astrocytes and, subsequently, microglia, where the regulated release of ATP as a result of partial lysosome exocytosis is important for the propagation of Ca2+ waves between neighboring cells and cell migration (Zhang et al., 2007; Dou et al., 2012). More recently, the group of Xianping Dong showed that lysosomes in a variety of cell lines, including HEK293 and COS1 cells, contain abundant ATP (Cao et al., 2014). They identified the transporter responsible for ATP accumulation in lysosomes as the vesicular nucleotide transporter (VNUT), otherwise known as solute carrier family 17 member 9 (SLC17A9; Cao et al., 2014). VNUT is responsible for ATP transport across secretory vesicle membranes in a variety of cells, including chromaffin cells and pancreatic acinar cells (Haanes et al., 2014). Cao et al. (2014) identified that VNUT is highly enriched in lysosome membranes and later showed that the energy to drive VNUT-mediated ATP uptake into lysosomes is the vesicular membrane potential established by the activation of lysosomal V-ATPases (Zhong et al., 2016). In their latest paper, Fois et al. (2018) show that VNUT similarly mediates ATP uptake into LBs and that dissipation of the LB membrane potential not only prevents uptake of ATP into LBs but also prevents its release upon stimulation of LB exocytosis. Knocking down the expression of VNUT in ATII cells, or pharmacological inhibition of VNUT activity, inhibits ATP release upon LB exocytosis. Importantly, it also completely ablates FACE, demonstrating the critical role of ATP from LBs in the activation of LB P2X4 receptors.

The role of P2X4 in the secretion of pulmonary surfactant clearly demonstrates the functional importance of these lysosomal receptors in ATII cells. The delivery of both the ion channel and its ligand to the surface of the cell upon LB exocytosis ensures precise signaling that is restricted to the fused LB. The question remains whether or not intracellular P2X4 receptors in ATII cells, or in the endolysosomes of other cells, can become activated and contribute to lysosome-derived Ca2+ signals and membrane traffic before their delivery to the plasma membrane. Lysosomal Ca2+ may come from endocytosis or by uptake from the cytosol in a manner dependent on pH, although in placental mammals the transporter responsible has not yet been identified (Xiong and Zhu, 2016). Lysosome-derived Ca2+ signals are not only required for lysosome fusion with other organelles and with the plasma membrane, but also play an important role in gene transcription and the regulation of autophagy. Arguably the best characterized of the lysosomal Ca2+ channels is TRPML1, which belongs to the large family of transient receptor potential ion channels (Xiong and Zhu, 2016). Mutations in the gene encoding TRPML1 lead to mucolipidosis type IV disease, which is characterized by defects in endolysosomal membrane traffic and enlarged lysosomes. In contrast, there are no known mutations in P2X4 that are associated with lysosomal disease, and mice with genetic ablation of P2X4 do not show gross lysosome abnormalities. Also, TRPML1 is activated by acidic pH, in contrast to the pH regulation of P2X4. Perhaps the best evidence for a functional role for intracellular P2X4 receptors is the study by Cao et al. (2015), which showed that either overexpression of P2X4 or pharmacological manipulation of lysosome pH promotes P2X4-dependent homotypic lysosome fusion and the generation of enlarged vacuolar lysosomes. This process is dependent on calmodulin, which associates with P2X4 in a Ca2+-dependent manner. Interestingly, P2X4-mediated enlargement of lysosomes can be recovered by either activation or overexpression of TRPML1, which promotes lysosome fission by a process that is also dependent on calmodulin (Cao et al., 2017).

The conditions under which endolysosomal P2X4 receptors become activated without either pharmacological manipulation of lysosomal pH or fusion with the plasma membrane remain unclear. Relevant to this is the recent demonstration that lysosomes represent a heterogeneous population of vesicles with differing luminal pH values (Bright et al., 2016). The fusion of lysosomes with late endosomes generates acidic endolysosomes with high cathepsin activity, whereas the terminal storage lysosomes are reportedly not acidic and contain inactive hydrolases. Similarly, peripheral lysosomes, which are likely to be those that undergo exocytosis, are less acidic than juxtanuclear ones by virtue of reduced V-ATPase activity and an increase in proton leak (Johnson et al., 2016). Therefore, the precise location of the endolysosomal P2X4 receptors will determine their regulation and involvement in the release of Ca2+ and recruitment of calmodulin. Lysosomes in cancer cells have altered function and are more secretory, which is thought to contribute to enhanced cancer cell invasiveness and metastases. Activation of the P2X7 receptor promotes lysosome exocytosis, cathepsin secretion, and an increase in the breakdown of the extracellular matrix in a highly invasive breast cancer cell line (Jelassi et al., 2011). In microglia cells, activation of P2X7 triggers lysosome alkalinization and increased exocytosis of autophagolysosomes (Takenouchi et al., 2009). It will be interesting to determine whether functional cross talk between lysosomal P2X4 and plasma membrane P2X7 receptors plays a role in driving these processes.

Acknowledgments

The author declares no competing financial interests.

Kenton J. Swartz served as editor.

References

References
Bobanovic
,
L.K.
,
S.J.
Royle
, and
R.D.
Murrell-Lagnado
.
2002
.
P2X receptor trafficking in neurons is subunit specific
.
J. Neurosci.
22
:
4814
4824
.
Bright
,
N.A.
,
L.J.
Davis
, and
J.P.
Luzio
.
2016
.
Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity
.
Curr. Biol.
26
:
2233
2245
.
Cao
,
Q.
,
K.
Zhao
,
X.Z.
Zhong
,
Y.
Zou
,
H.
Yu
,
P.
Huang
,
T.L.
Xu
, and
X.P.
Dong
.
2014
.
SLC17A9 protein functions as a lysosomal ATP transporter and regulates cell viability
.
J. Biol. Chem.
289
:
23189
23199
.
Cao
,
Q.
,
X.Z.
Zhong
,
Y.
Zou
,
R.
Murrell-Lagnado
,
M.X.
Zhu
, and
X.P.
Dong
.
2015
.
Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion
.
J. Cell Biol.
209
:
879
894
.
Cao
,
Q.
,
Y.
Yang
,
X.Z.
Zhong
, and
X.P.
Dong
.
2017
.
The lysosomal Ca2+ release channel TRPML1 regulates lysosome size by activating calmodulin
.
J. Biol. Chem.
292
:
8424
8435
.
Dou
,
Y.
,
H.J.
Wu
,
H.Q.
Li
,
S.
Qin
,
Y.E.
Wang
,
J.
Li
,
H.F.
Lou
,
Z.
Chen
,
X.M.
Li
,
Q.M.
Luo
, and
S.
Duan
.
2012
.
Microglial migration mediated by ATP-induced ATP release from lysosomes
.
Cell Res.
22
:
1022
1033
.
Fois
,
G.
,
V.E.
Winkelmann
,
L.
Bareis
,
L.
Staudenmaier
,
E.
Hecht
,
C.
Ziller
,
K.
Ehinger
,
J.
Schymeinsky
,
C.
Kranz
, and
M.
Frick
.
2018
.
ATP is stored in lamellar bodies to activate vesicular P2X4 in an autocrine fashion upon exocytosis
.
J. Gen. Physiol.
Haanes
,
K.A.
,
J.M.
Kowal
,
G.
Arpino
,
S.C.
Lange
,
Y.
Moriyama
,
P.A.
Pedersen
, and
I.
Novak
.
2014
.
Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells
.
Purinergic Signal.
10
:
431
440
.
Huang
,
P.
,
Y.
Zou
,
X.Z.
Zhong
,
Q.
Cao
,
K.
Zhao
,
M.X.
Zhu
,
R.
Murrell-Lagnado
, and
X.P.
Dong
.
2014
.
P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH
.
J. Biol. Chem.
289
:
17658
17667
.
Jelassi
,
B.
,
A.
Chantôme
,
F.
Alcaraz-Pérez
,
A.
Baroja-Mazo
,
M.L.
Cayuela
,
P.
Pelegrin
,
A.
Surprenant
, and
S.
Roger
.
2011
.
P2X(7) receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness
.
Oncogene.
30
:
2108
2122
.
Johnson
,
D.E.
,
P.
Ostrowski
,
V.
Jaumouillé
, and
S.
Grinstein
.
2016
.
The position of lysosomes within the cell determines their luminal pH
.
J. Cell Biol.
212
:
677
692
.
Miklavc
,
P.
,
N.
Mair
,
O.H.
Wittekindt
,
T.
Haller
,
P.
Dietl
,
E.
Felder
,
M.
Timmler
, and
M.
Frick
.
2011
.
Fusion-activated Ca2+ entry via vesicular P2X4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes
.
Proc. Natl. Acad. Sci. USA.
108
:
14503
14508
.
Miklavc
,
P.
,
E.
Hecht
,
N.
Hobi
,
O.H.
Wittekindt
,
P.
Dietl
,
C.
Kranz
, and
M.
Frick
.
2012
.
Actin coating and compression of fused secretory vesicles are essential for surfactant secretion--a role for Rho, formins and myosin II
.
J. Cell Sci.
125
:
2765
2774
.
Qureshi
,
O.S.
,
A.
Paramasivam
,
J.C.
Yu
, and
R.D.
Murrell-Lagnado
.
2007
.
Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis
.
J. Cell Sci.
120
:
3838
3849
.
Ridsdale
,
R.
,
C.L.
Na
,
Y.
Xu
,
K.D.
Greis
, and
T.
Weaver
.
2011
.
Comparative proteomic analysis of lung lamellar bodies and lysosome-related organelles
.
PLoS One.
6
:
e16482
.
Stokes
,
L.
,
J.A.
Layhadi
,
L.
Bibic
,
K.
Dhuna
, and
S.J.
Fountain
.
2017
.
P2X4 Receptor Function in the Nervous System and Current Breakthroughs in Pharmacology
.
Front. Pharmacol.
8
:
291
.
Suurväli
,
J.
,
P.
Boudinot
,
J.
Kanellopoulos
, and
S.
Rüütel Boudinot
.
2017
.
P2X4: A fast and sensitive purinergic receptor
.
Biomed. J.
40
:
245
256
.
Takenouchi
,
T.
,
M.
Fujita
,
S.
Sugama
,
H.
Kitani
, and
M.
Hashimoto
.
2009
.
The role of the P2X7 receptor signaling pathway for the release of autolysosomes in microglial cells
.
Autophagy.
5
:
723
724
.
Thompson
,
K.E.
,
J.P.
Korbmacher
,
E.
Hecht
,
N.
Hobi
,
O.H.
Wittekindt
,
P.
Dietl
,
C.
Kranz
, and
M.
Frick
.
2013
.
Fusion-activated cation entry (FACE) via P2X4 couples surfactant secretion and alveolar fluid transport
.
FASEB J.
27
:
1772
1783
.
Xiong
,
J.
, and
M.X.
Zhu
.
2016
.
Regulation of lysosomal ion homeostasis by channels and transporters
.
Sci. China Life Sci.
59
:
777
791
.
Zhang
,
Z.
,
G.
Chen
,
W.
Zhou
,
A.
Song
,
T.
Xu
,
Q.
Luo
,
W.
Wang
,
X.S.
Gu
, and
S.
Duan
.
2007
.
Regulated ATP release from astrocytes through lysosome exocytosis
.
Nat. Cell Biol.
9
:
945
953
.
Zhong
,
X.Z.
,
Q.
Cao
,
X.
Sun
, and
X.P.
Dong
.
2016
.
Activation of lysosomal P2X4 by ATP transported into lysosomes via VNUT/SLC17A9 using V-ATPase generated voltage gradient as the driving force
.
J. Physiol.
594
:
4253
4266
.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).