P2X receptors are a group of homo/hetero-trimeric membrane protein complexes with
an integral ion channel that opens upon extracellular adenosine triphosphate (ATP)
binding (North, 2002; Khakh and North, 2006). There are seven P2X subunits (P2X1-P2X7),
all having a membrane topology of cytosolic N- and C-termini, and two transmembrane
segments (TM1 and TM2) connected by a large extracellular domain (Figure 1A; Jiang
L.-H. et al., 2013). During application of ATP for a few seconds, P2X receptors function
as classical ligand-gated ion channels selectively permeable to small physiological
cations such as Ca2+, Na+, and K+, with the exception of the human P2X5 receptor which
exhibits significant Cl− permeability (Bo et al., 2003). Site-directed mutagenesis
and functional studies of mammalian P2X receptors, in addition to the determination
of the crystal structures of zebrafish P2X4 receptors in the apo, closed state and
ATP-bound, open state, have defined the structural basis for ATP binding, ion permeation
and channel gating (Kawate et al., 2009; Browne et al., 2010; Hattori and Gouaux,
2012; Jiang L.-H. et al., 2013; Jiang R. et al., 2013). Three ATP-binding pockets
are located at the subunit interfaces (Figure 1B), each consisting of highly conserved
residues from two adjacent subunits. Occupation of these sites by ATP or its synthetic
analog agonists induces conformational changes of the extracellular domain which open
the ion-permeating pathway formed by three TM2s (Figures 1C,D). The narrowest part
of the ion-permeating pathway or the physical gate is provided by A347 and L351 in
the crystal structures of zebrafish P2X4 receptor (Hattori and Gouaux, 2012) or the
corresponding residues S342 and L346 in the structural models of rat and human P2X7
receptors (Figure 1D) (Bradley et al., 2011; Browne et al., 2013; Jiang L.-H. et al.,
2013).
Figure 1
Structural models of the human P2X7 ion channel in the closed and open state. (A,B)
The structural models of the trimeric human P2X7 receptor in the closed (A) and open
states (B), viewed parallel to the plasma membrane. They are generated based on the
crystal structures of zebrafish P2X4 receptor (4DW0 and 4DW1 respectively). Each subunit
is shown in a different color. Three ATP molecules shown in space filling representation
bind to the three inter-subunit interfaces (B). (C,D) The transmembrane ion-permeating
pathway in the structural models of the human P2X7 receptor in the closed (C) and
open states (D), viewed from the intracellular side of the membrane. The three TM1
helices are located at the periphery and the three TM2 helices in the center form
the ion-conducting pathway. Ser342 and Leu346 from each of the three subunits provide
the narrow part of the ion-conducting pathway in the open state (D).
It is well known that extended application of ATP to activate the P2X receptors for
tens of seconds or minutes induces a remarkable increase in membrane permeability
to large molecules of up to 900 Daltons, a phenotype often referred to as formation
of large pores. This was originally documented in immune cells about three decades
ago; ATP permeablized cell membranes to nucleotides (Cockcroft and Gomperts, 1979)
and the cationic fluorescent dye ethidium in mast cells (Gomperts, 1983), or the anionic
organic dyes lucifer yellow and carboxyfluoresceine in mast cells and macrophages
(Bennett et al., 1981; Steinberg and Silverstein, 1987). These immune cells express
the formerly named P2Z receptor, which is now known as P2X7 receptor, and heterologous
expression of P2X7 receptors conferred ATP-induced large pore formation (Surprenant
et al., 1996). Such large pore formation has also been observed during sustained activation
of other P2X receptors including P2X2, P2X2/3, P2X2/5, and P2X4 receptors (Khakh et
al., 1999; Virginio et al., 1999a,b; Compan et al., 2012). Substantial efforts have
been devoted to understanding P2X receptor-dependent large pore formation, but it
has been difficult to interpret in a unified mechanism all results from studies examining
different receptors in different cell types with different receptor expression levels.
Two distinctive mechanisms or hypotheses have been proposed (North, 2002; Pelegrín,
2011; Jiang L.-H. et al., 2013). The first is that persistent activation of P2X receptors
induces dilation of the small ion-permeating pathway. The second, alternative mechanism
is that a separate membrane protein interacts with the P2X receptor and forms the
large pores upon activation of P2X receptors. For example, pannexin-1 was shown to
form large pores associated with activation of the P2X7 receptor (Pelegrin and Surprenant,
2006).
Two experimental approaches are commonly used to study P2X receptor-dependent large
pore formation. The biophysical approach is to patch-clamp record agonist-induced
currents with Na+ and N-methyl-D-glucamine (NMDG+) being the cation in intracellular
and extracellular solutions, respectively (Surprenant et al., 1996; Khakh et al.,
1999; Virginio et al., 1999a,b). Under such bi-ionic conditions, if the cell membrane
is held at a negative potential, activation of P2X receptors induces initially outward
currents which decline in amplitude as the receptor activation continues. Within tens
of seconds these outwards currents change into inward currents. The current reversal
potential exhibits a progressive shift toward the less negative direction (example
recordings see Surprenant et al., 1996; Khakh et al., 1999; Virginio et al., 1999a,b;
Bo et al., 2003; Jiang et al., 2005). If one assumes the intracellular and extracellular
cation concentrations remain unchanged during patch-clamp recording, the shift in
the current reversal potential can be interpreted as a result of an increase in the
NMDG+ permeability of the cell membrane, namely, the open ion channel is poorly permeable
to NMDG+ at the beginning of receptor activation but significantly increase its NMDG+
permeability as the receptor activation continues. Such an interpretation has led
to the pore dilation hypothesis (Virginio et al., 1999a; North, 2002). In addition
to the P2X receptors, this biophysical approach has been used to show increases in
the permeability during activation of other ion channels such as TRPV1 (Chung et al.,
2008; Samways et al., 2008; Munns et al., 2015) and TRPA1 (Chen et al., 2009). The
second method to study large pore formation is to use fluorescence microscopy or a
fluorescence detection system to monitor agonist-induced intracellular accumulation
of fluorescence dyes such as YO-PRO-1 and ethidium, or alternatively agonist-induced
progressive loss of preloaded fluorescence dyes such as calcein (example recordings
see Surprenant et al., 1996; Virginio et al., 1999a,b; Jiang et al., 2005; Sorge et
al., 2012). Measurements of dye uptake (or loss) are often made in more physiological
solutions containing micromolar concentrations of fluorescent dye and, by and large,
avoid the complications associated with complete removal of extracellular physiological
cations. One well-documented example of such complications is that the P2X7 and P2X2/5
ion channels activated in extracellular NMDG+-containing solutions were somehow trapped
in an open state and did not return to the closed state even minutes after agonist
application was discontinued (Jiang et al., 2005; Yan et al., 2008; Compan et al.,
2012). Therefore, the findings from measurements of fluorescence dye uptake are of
much more biological relevance. The amplitude and rate of fluorescence dye uptake
are grossly indicative of large pore formation (e.g., Roger et al., 2010; Browne et
al., 2013).
It was assumed in previous studies, despite not always being stated explicitly, that
P2X receptor-dependent large pores serve as the common pathway permeating NMDG+ and
fluorescent dye uptake. However, this was challenged in a previous study examining
the rat P2X7 receptor heterologously expressed in human embryonic kidney (HEK) 293
cells (Jiang et al., 2005). The study showed that sustained activation of P2X7 receptor
in extracellular Na+-containing solutions induced robust YO-PRO-1 dye uptake but,
surprisingly, no increase in the PNMDG/PNa. In addition, the study found that removal
of a cysteine-rich microdomain in the proximal part of the intracellular C-terminus
almost completely abolished agonist-induced reversal potential shift under bi-ionic
conditions without compromising agonist-induced YO-PRO-1 uptake. In fact, as compared
to the wild-type receptor, expression of the deletion mutant receptor resulted in
higher YO-PRO-1 uptake in both Na+-containing and NMDG+-containing solutions (Jiang
et al., 2005). These two independent lines of evidence strongly argue against the
idea that a same molecular mechanism is used to mediate the entry of both NMDG+ and
YO-PRO-1 into the cell. In HEK293 cells heterologously expressing the rat P2X2 receptor,
a recent study has found that ATP activation of the P2X2 receptor in extracellular
Na+-containing solutions induced no increase in the PNMDG/PNa (Li et al., 2015). The
study has elegantly introduced a reservoir model to support the notion that the reversal
potential shift simply arises from substantial reduction in the intracellular Na+
concentration and increase in the intracellular NMDG+ concentration during prolonged
P2X2 ion channel opening. In their model, the P2X2 ion channel is NMDG+-permeable,
albeit with the PNMDG/PNa of 0.05, but there is no need for an increase in the NMDG+
permeability, in other words, no pore dilation! The study has demonstrated that the
open P2X2 ion channel permeates NMDG+ as quickly as small cations like Na+, but not
as easily as the latter ions. The P2X7 open ion channels also exhibit extremely low,
if any, NMDG+ permeability (PNMDG/PNa ~ 0.03-0.04; Virginio et al., 1999a; Jiang et
al., 2005). Structural modeling based on the open state structure of zebrafish P2X4
receptor (Hattori and Gouaux, 2012) positions the Cα atoms of three S342 residues
in the physical gate of the ion-permeating pathway as being 6.4 Å from the central
axis in both rat and human P2X7 receptors (Browne et al., 2013; Jiang L.-H. et al.,
2013; Figure 1D). NMDG+ has a size of 6 Å × 6 Å × 12.5Å and therefore, as proposed
in a recent study (Browne et al., 2013), the P2X7 open ion channels may be sufficiently
wide to permeate NMDG+.
The commonly used fluorescent dyes are, however, considerably larger in size than
NMDG+, for example YO-PRO-1 (7 Å × 8 Å × 19Å) and ethidium (6.5 Å × 11 Å × 13Å) (Browne
et al., 2013). How do the fluorescent dyes come across the cell membrane, also through
the ion-permeating pathway? Previous studies showed YO-PRO-1 uptake following activation
of P2X2, P2X2/3, and P2X4 receptors (Khakh et al., 1999; Virginio et al., 1999b).
The open ion channels of these receptors, if permitting passage of YO-PRO-1, have
to open much more widely than the above-mentioned ion-permeating pathway revealed
in the open state structure of zebrafish P2X4 receptor (Hattori and Gouaux, 2012).
Such a possibility remains to be tested. A recent study has investigated whether the
rat P2X7 ion channel was able to permeate large molecules, including the cationic
fluorescent dyes YO-PRO-1 and ethidium, the anionic fluorescent dye fluorescein isothiocyanate
(FITC; 8.5 Å × 11 Å × 14.5Å), and neutral cysteine-modifying 2-aminoethyl methanethiosulfonate
(MTSEA; 5 Å × 5 Å × 10Å), MTSEA-biotin (7.5 Å × 8 Å × 18.5Å) and MTS-rhodamine (9
Å × 14 Å × 16.5 Å) (Browne et al., 2013). ATP-induced ionic currents and YO-PRO-1
uptake both strongly depend on membrane potential, the driving force for movement
of charged molecules. ATP also induced uptake of ethidium and FITC in a correlating
fashion even though these two dyes bear opposite charges. ATP-induced ethidium uptake
was reduced and by contrast ATP-induced FITC uptake was increased by membrane depolarization.
Furthermore, introduction of a positive charge by T348K mutation or neutralization
of a negative charge by D352N in the small ion-permeating pathway resulted in a decrease
in ATP-induced ethidium uptake but an increase in ATP-induced FITC uptake. Finally,
MTSEA-biotin and MTS-rhodamine as well as MTSEA readily modified cysteine replacing
G345, a position internal to the narrowest part of the P2X7 open ion channel, and
inhibited ATP-induced ionic currents and ethidium uptake. These results provide direct
evidence to demonstrate that the rat P2X7 open ion channel can permeate large molecules.
However, to accomplish this, the open ion channels need to be a minimum of 14 Å wide.
This is noticeably wider than the ion-permeating pathway in the open state models
of rat and human P2X7 receptors, supporting the notion that the open ion channel dilates
(Virginio et al., 1999a; Browne et al., 2013). Structural determination of the ion-permeating
pathway of a mammalian P2X receptor in the open state will provide the key answer
to whether or how the open ion channel allows passage of fluorescent dyes.
In parallel with these efforts to understand P2X receptor-dependent large pore formation,
studies have accumulated evidence to show the importance of such receptor functionality.
For example, P2X7 receptor-dependent large pore formation has been identified as a
crucial factor associated with disease conditions such as chronic pain (Sorge et al.,
2012), osteoporosis (Syberg et al., 2012) and geographic atrophy (Fowler et al., 2014).
Furthermore, preferential inhibition of P2X7 receptor-dependent large pore formation
has been proposed in a recent study to be the molecular mechanism underpinning the
anti-inflammatory activity of nucleoside reverse transcriptase inhibitors, a class
of clinically proven drugs treating HIV (Fowler et al., 2014). Selective targeting
of P2X7 receptor-dependent large pore formation appears a promising and novel pharmacological
intervention (Jiang, 2015). Therefore, it becomes increasingly interesting and important
to gain a better mechanistic insight into large pore formation after activation of
P2X receptors, in particular P2X7 receptors.
Author contributions
LW, L-HJ and DL led the discussion; EC contributed to the discussion and generated
the structural models. L-HJ wrote the manuscript, and all authors commented the manuscript.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.