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The Journal of General Physiology logoLink to The Journal of General Physiology
. 2023 May 18;155(7):e202213317. doi: 10.1085/jgp.202213317

Extracellular histone proteins activate P2XR7 channel current

Rua’a Al-Aqtash 1, Maxwell S Ross 1, Daniel M Collier 1,✉
PMCID: PMC10200710  PMID: 37199689

Histone proteins are known to be elevated in circulation where they contribute to vascular dysfunction. However, many details regarding the mechanism of action are unknown. Here, the authors show that P2XR7 channel current is activated by extracellularly applied histone proteins in a heterologous expression system.

Abstract

Extracellular histone proteins are elevated in circulation after injury or activation of the innate immune response. In resistance-size arteries, extracellular histone proteins increased endothelial cell (EC) Ca2+ influx and propidium iodide (PI) labeling, but paradoxically decreased vasodilation. These observations could be explained by the activation of an EC resident non-selective cation channel. We tested the hypothesis that the ionotropic purinergic receptor 7 (P2XR7), a non-selective cation channel associated with cationic dye uptake, is activated by histone proteins. We expressed mouse P2XR7 (C57BL/6J variant 451L) in heterologous cells and measured inward cation current using two-electrode voltage clamp (TEVC). Cells expressing mouse P2XR7 had robust ATP- and histone-evoked inward cation currents. ATP- and histone-evoked currents reversed approximately at the same potential. Current decay with agonist removal was slower for histone-evoked than ATP- or BzATP-evoked currents. As with ATP-evoked P2XR7 currents, histone-evoked currents were inhibited by non-selective P2XR7 antagonists (Suramin, PPADS, and TNP-ATP). Selective P2XR7 antagonists, AZ10606120, A438079, GW791343, and AZ11645373, inhibited ATP-evoked P2XR7 currents but did not inhibit histone-evoked P2XR7 currents. As previously reported with ATP-evoked currents, histone-evoked P2XR7 currents were also increased in conditions of low extracellular Ca2+. These data demonstrate that P2XR7 is necessary and sufficient for histone-evoked inward cation currents in a heterologous expression system. These results provide insight into a new allosteric mechanism of P2XR7 activation by histone proteins.

Introduction

Histone octamer core proteins (H2a, H2b, H3, and H4) are most well known in their role as DNA-binding proteins that facilitate chromatin formation. However, levels of histone proteins are elevated in circulation following cellular damage or activation of the innate immune response. Activation of the innate immune response causes neutrophil recruitment and production of histone-rich neutrophil extracellular traps (NETs) via NETosis as reviewed by Remijsen et al. (2011). NETs and histone proteins have been shown to be elevated clinically after traumatic injury, with acute respiratory syndrome (ARDS), or with infection such as COVID-19 (Albert et al., 2018; Russell et al., 2018; Leppkes et al., 2020; Wang et al., 2020; Zuo et al., 2020). In a murine model of mild traumatic brain injury, neutrophil recruitment to the lungs is increased through activation of the innate immune response (Vaickus et al., 2019). Murine models of sepsis and traumatic injury showed an association of elevated histone proteins in circulation with vascular dysfunction and death (Xu et al., 2009). Plasma histone levels correlate with ARDS and trauma severity and range from ∼2 μg/ml in healthy control patients to more than ∼10–100 μg/ml depending on severity (Abrams et al., 2013; Lv et al., 2017).

It has been shown that histone proteins, at 10 μg/ml, cause localized Ca2+ influx and propidium iodide (PI) uptake in the endothelium of resistance-size mouse mesenteric arteries, yet paradoxically inhibit vasodilation to hyperpolarizing and well-described vasodilatory Ca2+-dependent pathways (Collier et al., 2019). However, the mechanism of histone-induced Ca2+ influx is unknown. Elevated PI uptake suggested that one mechanism could be endothelial cell (EC) death. Cationic DNA-binding dyes, such as PI or the yellow-shifted YO-PRO analog, can permeate dead cells but not live cells. Thus, dye uptake is commonly interpreted as a marker of cell death. However, dye uptake in live cells can be achieved in low Ca2+ conditions using the reversible permeabilization technique that relies on prolonged activation of P2XR7 in low Ca2+ conditions (Morgan and Morgan, 1982, 1984; Innocenti et al., 2004). P2XR7 activation may directly or indirectly, through recruitment of other proteins, increase dye uptake. P2XR7 was necessary and sufficient for dye uptake in liposomes and dependent on lipid composition (Karasawa et al., 2017). However, others have demonstrated that P2XR7 activation may recruit other proteins to facilitate dye uptake such as pannexin (Pelegrin and Surprenant, 2006). Dye uptake is, therefore, often used as an indirect measure of P2XR7 activity.

The ionotropic purinergic receptor (P2XR) family is composed of seven hetero- and homotrimeric non-selective cation channels (P2XR1–7). All members have relatively short intracellular N-termini, large extracellular receptor domains, and variable length intracellular C-termini. One of the key limitations to understanding the physiological and pathological roles of P2XR channels in native tissue is the lack of truly selective pharmacology. All P2XRs are activated by extracellular ATP but differ in ATP affinity and desensitization kinetics. The structures of multiple P2XRs in putative closed, open (ATP bound), and antagonist-bound states have been solved by crystallography and cryo-EM (Gonzales et al., 2009; Kawate et al., 2009; Karasawa and Kawate, 2016; Mansoor et al., 2016; McCarthy et al., 2019). P2XR7 differs from other P2XRs in several key aspects. P2XR7 requires very high concentrations of ATP (millimolar) and does not desensitize in the continued presence of ATP, facilitates permeation of large cationic dyes, is regulated by membrane lipid interactions, and has C-terminal binding domains not found in other P2XRs (North and Surprenant, 2000; North, 2002; Karasawa et al., 2017; McCarthy et al., 2019). These unique functional traits, along with relatively poor sensitivity to the putative endogenous agonist, ATP, raise the interesting possibility that there may be alternative physiologic agonists for P2XR7 channel current.

Precedence for activation of P2XR7 by non-nucleotide agonists exists in the literature and has been reviewed by Di Virgilio et al. (2017). Of particular relevance to our work, the human cathelicidin peptide (LL-37), a 37 aa polybasic peptide, has been shown to activate P2XR7-mediated dye uptake (Tomasinsig et al., 2008). This supports the possibility that histone proteins could be an additional non-nucleotide agonist of P2XR7 channels. Activation of P2XR7, a non-selective cation channel, would depolarize the endothelium and short-circuit canonical endothelium-dependent hyperpolarizing pathways. Thus, EC-resident P2XR7 activation could be the mechanism of loss of vasodilation with histone exposure. Herein, we test the hypothesis that extracellular histone proteins are activators of P2XR7 channel currents in a controlled, heterologous expression, system.

Materials and methods

DNA constructs

cDNA for mouse and human P2XR7 was obtained from Origene (MR227216 and RC222143; Origene Technologies). Primers for mutagenesis and sequencing were designed with SnapGene (v5.1; GSL Biotech LLC). Mutations were generated by site-directed mutagenesis (QuickChangeII XL; Stratagene). All constructs were fully sequenced to verify construct identity (Azenta).

Reagents

All chemicals to make the solutions were obtained from Sigma-Aldrich. Unfractionated histone proteins, consisting of a mixture of histone core subunits (Collier et al., 2019), were obtained from Sigma-Aldrich (#10223565001; Millipore Sigma). Pure, recombinant human histone H2a, H2b, H3, and H4 were obtained from Abcam (ab200295, ab198637, ab198757, ab198115; Abcam). Proteinase K (Pro K) was obtained from New England Biolabs (#P8107S; NEB).

Expression and whole-cell electrophysiology in Xenopus oocytes

Defolliculated, injection-ready, oocytes were obtained from Xenopus1 or EcoCyte. Oocyte nuclei were injected with plasmid cDNAs encoding human P2XR7 (NCBI accession no. NP_002553.3) mouse (c57) wild-type (451L, MGI:1339957; homologous to NP_002553.3) or mutant P2XR7 channels as indicated in figure legends (0.06 μg/μl per cell). Cells were incubated at 18°C in modified Barth’s saline (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3, 10 mM HEPES, 50 μg/ml gentamycin sulfate, 10 μg/ml sodium penicillin, and 10 μg/ml streptomycin sulfate, pH adjusted to 7.4 with 1 M NaOH) for 20–24 h prior to study. Oocytes were voltage-clamped (two-electrode voltage clamp) and currents were amplified with an Oocyte Clamp OC-725C (Warner Instruments). Currents were digitized with an InstruTECH data acquisition system (#895035; Instrutech LIH 8+8, Warner Instruments) and recorded using ChartMaster (V2x90.5, HEKA, Harvard Biosciences). Unless otherwise noted, recordings were done at −60 mV in a 116 mM NaCl modified Ca2+ Ringer’s solution (116 mM NaCl, 2 mM KCl, 2.0 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH adjusted to 7.4 with NaOH). Standard oocyte Ringer’s (low Ca2+) was used to measure the effect of Ca2+ on histone and ATP-evoked currents (116 mM NaCl, 2 mM KCl, 0.4 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH adjusted to 7.4 with NaOH). Cells were screened for P2XR7 expression by applying 0.6–1.0 mM ATP (as indicated in figure legends). Uninjected oocytes were used as negative controls for histone- and ATP-evoked P2XR7 currents. In some experiments as indicated, histone-evoked currents are normalized relative to ATP-evoked currents from the same cell account for cell-to-cell differences in total P2XR7 expression.

Statistical analysis

All values were provided as mean ± SEM. The data fitting and statistical analysis were performed with the latest version of Prism (GraphPad 9.4.1). Statistical significance was determined using a Student’s t test or ANOVA with corrections for multiple comparisons as appropriate. Sample size (n, biological replicates in number of animals and cells), statistical test used, and results (P values) are reported in the figure legends.

Results

Purified histone octamer proteins activate P2XR7 currents

To determine whether histone proteins can activate the P2XR7 channel, we expressed mouse P2XR7 (451L variant) in Xenopus oocytes. Oocytes were injected with plasmid cDNA encoding P2XR7. Uninjected, non-P2XR7 expressing, cells were used as negative control. Cells were screened for P2XR7 channel activity with 600 µM ATP. Cells were washed to remove ATP and allowed to recover for at least 30 s prior to application of 10 µg/ml histone, a concentration at the low end of the range of histones reported in circulation after trauma or ARDS and sufficient to cause robust EC Ca2+ influx and vascular dysfunction in resistance-size mesenteric arteries (Abrams et al., 2013; Lv et al., 2017; Collier et al., 2019). Histone protein application induced large inward cation currents in oocytes expressing mouse (“m.”, B) or human (“h.”, C) P2XR7 while neither ATP nor histone application produced current in control, non-P2XR7 expressing, cells (Fig. 1, A–C). To estimate the relative agonist affinity, we fit the rate of current decay during agonist wash to a single exponential (off-rate, “tau-off”). BzATP is a more potent activator of P2XR7 than ATP. P2XR7 is activated by micromolar levels of BzATP and millimolar levels of ATP. All three agonists induced large, sustained, inward currents; however, the off-rates for both ATP and BzATP were significantly faster than that for histone-evoked current (Fig. 1 D). These data demonstrate that histone-evoked current is dependent on P2XR7 expression and produces currents with slower reversal kinetics than ATP- or BzATP-evoked P2XR7 currents.

Figure 1.

Figure 1.

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Purified histone octamer proteins activate P2XR7 currents. (A) Representative traces of current versus time recorded from a Xenopus oocyte 24–48 h after nuclear injection of plasmid cDNA encoding mouse P2XR7. Cells were held at −60 mV. 1 s voltage ramps from −60 to 40 mV were performed at baseline and in the presence of 600 μM ATP or 10 μg/ml histone protein in high Ca2+ Ringer’s solution. (B and C) Average whole-cell current at −60 mV in oocytes 24–48 h after nuclear injection of mouse (B, “m.”) or human (C, “h.”) P2XR7 cDNA in response to 600 μM ATP, 50 μM BzATP, or 10 μg/ml histones (uninjected cells, “Ctrl”; Ave ± SEM, n = 3–9 animals, 8–28 cells). (D) ATP-, BzATP-, and histone-evoked m.P2XR7 current decay with agonist removal estimated by fitting data from A (highlighted in gray) to a single exponential (Ave ± SEM, n = 3 animals, 3–10 cells, * P = 0.0016 histone vs. BzATP, * P < 0.0001 histone vs. ATP). (E and F) Representative current–voltage relationships measured once peak sustained currents were achieved in A (n = 3 animals, 10 cells). (G and H) Average reversal potential (G) and whole-cell conductance (H) for ATP- and histone-evoked currents in oocytes expressing P2XR7 calculated from voltage ramps in E and F (Ave ± SEM, n = 3 animals, 10 cells). (I and J) average ATP- or histone-evoked inward current measured at different holding potentials (Ave ± SEM, some error bars are smaller than their marker, n = 2–3 animals, 8–10 cells, reversal = −24 mV for ATP and −39 mV for histone). Gray line represents best fit linear regression. (J) Histone-evoked currents were normalized to ATP-evoked currents from the same cell (Ave ± SEM, some error bars are smaller than their marker, n = 2–3 animals, 8–10 cells, reversal = −35 mV). (K) Average whole-cell current in P2XR7-expressing cells in response to histone concentrations from 1 to 10 μg/ml and in response to boiled and Pro K digested histone protein at 10 μg/ml (Pro K–treated histones also boiled to inactivate Pro K, Ave ± SEM, n = 3–9 animals, 8–20 cells, * P < 0.0001 by non-parametric one-way ANOVA).

It has been proposed that histone proteins act by directly interacting with the plasma membrane and create a pore or cause membrane disruption as reviewed by Silk et al. (2017). If histone proteins increase whole-cell current independent of P2XR7 activation, we would expect a shift in the reversal potential of histone-evoked currents relative to ATP-evoked currents to reflect the activation of a P2XR7 channel-independent conduction pathway. We measured ATP- and histone-evoked reversal potentials in P2XR7 expressing cells. We applied voltage ramps from −60 to 40 mV after activation with ATP or histone proteins and estimated the reversal potential (Fig. 1, E–G). We observed no significant difference in the reversal potential of P2XR7 ATP- or histone-evoked currents (−28 and −27 mV, respectively; Fig. 1, A, and E–G). Voltage ramps from Fig. 1 A were fit using linear regression to calculate whole-cell conductance. Consistent with currents recorded at −60 mV, histone-evoked whole-cell conductance was slightly lower than ATP-evoked conductance (Fig. 1 H). These data, in combination with our observation that histone proteins only evoke currents in P2XR7 expressing cells (Fig. 1, B and C), suggest that histone proteins are not directly forming a transmembrane pore, as histone-evoked current is dependent on P2XR7 channel expression and retains basic P2XR7 current properties.

If histone proteins are binding P2XR7 channels near the membrane, we would predict the interaction to be voltage-dependent given the positive charge of histone proteins at neutral pH. Current–voltage relationships were ohmic for ATP-evoked currents (Fig. 1 E), which is known to bind near the apex of the extracellular domain, far from any influence of the electric field across the membrane (McCarthy et al., 2019). Current–voltage relationships are also ohmic for histone-evoked currents (Fig. 1 F). We measured the voltage dependence of P2XR7 channel activation by ATP and histones. ATP- and histone-evoked currents were recorded in P2XR7 expressing cells, as in Fig. 1 A, over a range of holding potentials from −100 to −40 mV (Fig. 1 I). The voltage dependence of activation was also well fit with simple linear regression (Fig. 1 I, gray lines). While there is a slight difference in reversal potential of these currents, they still reverse near −30 mV, as observed with voltage ramps applied after activation at −60 mV (Fig. 1, A and G). Some of this variability may be due to cell-to-cell variation in P2XR7 total expression or slight changes in intracellular ionic conditions with longer duration holding at different membrane potentials. We normalized histone-evoked currents to ATP-evoked currents from the same cell to reduce variability and fit the resulting ratio/voltage relationship using simple linear regression (Fig. 1 J). This relationship reverses at −35 mV, in closer agreement with voltage-ramps recorded in Fig. 1 A. These data demonstrate that histone proteins activate P2XR7 channel currents by a mechanism that is not voltage-dependent.

In previous work, we demonstrated a concentration-dependent increase in total histone-induced Ca2+ influx in the endothelium of en face resistance-size mesenteric arteries (Collier et al., 2019). We replicated these experiments in P2XR7 expressing cells. Consistent with our previous work, significant histone-evoked currents were observed in response to as little as 3 µg/ml histones (Fig. 1 K). We previously reported that boiling histone proteins was insufficient to eliminate activity (Collier et al., 2019). Histone proteins must be digested with Pro K to prevent Ca2+ influx (Collier et al., 2019). We see the same effect with histone-evoked P2XR7 currents. Enzymatic digestion of histone protein with Pro K (followed by heat inactivation of Pro K) prevented histone-evoked currents (Fig. 1 K). These findings provide evidence that intact histone protein subunits are necessary to activate P2XR7 channel currents.

Histone-evoked P2XR7 currents are inhibited by non-selective P2XR antagonists, but not inhibited by selective P2XR7 antagonists

To determine the efficacy of existing pharmacologic inhibitors of P2XR7 on histone-evoked currents, we measured histone-evoked currents in the presence of non-selective and selective P2XR antagonists. Application of Suramin (5 μM) inhibited both ATP- and histone-evoked currents in P2XR7 expressing cells (Fig. 2, A and B). Fig. 2 B shows average histone-evoked currents 30 s after application of vehicle (Ctrl), PPADS, TNP-ATP, or Suramin. All non-selective antagonists used significantly decreased histone-evoked currents in P2XR7 expressing cells (Fig. 2, A and B). Recent structural work resolved P2XR7 with selective antagonists bound. One of these compounds, GW791343 (GW), binds to an allosteric inhibitory site along the central axis of the extracellular domain, near to, but distinct from, the ATP binding site (Karasawa and Kawate, 2016). In this work, GW inhibition demonstrated the most promising properties—large inhibition and rapid and complete reversibility (Karasawa and Kawate, 2016). We hypothesized that GW might also inhibit histone-evoked P2XR7 currents. In our system, GW inhibits ATP-evoked P2XR7 currents to a similar extent as reported by Karasawa et al. (2017) (Fig. 2, C and F; Karasawa and Kawate, 2016). We verified other P2XR7 selective antagonists and found similar inhibition of ATP-evoked P2XR7 currents as reported in the literature (Fig. 2, D–H). However, GW did not inhibit histone-evoked P2XR7 currents (Fig. 2, I and L). Similarly, none of the potent P2XR7 selective antagonist tested inhibited histone-evoked P2XR7 currents (Fig. 2, J–N). Inhibition may be state-dependent. To test this, we verified P2XR7 expression with ATP, washed ATP away, applied antagonist, and activated P2XR7 currents with ATP or histone proteins in the continued presence of antagonist (Fig. 2, O–Q). We observed a similar trend as with applying antagonist to already opened channels. Pre-application of selective P2XR7 antagonist inhibited ATP-evoked P2XR7 currents, but had no effect on histone-evoked P2XR7 currents (Fig. 2, P and Q). This data demonstrate that some P2XR antagonists can successfully inhibit histone-evoked P2XR7 currents while selective allosteric P2XR7 antagonists have no effect on histone-evoked P2XR7 currents.

Figure 2.

Figure 2.

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Histone-evoked P2XR7 currents are inhibited by non-selective P2XR antagonists, but not inhibited by selective P2XR7 antagonists. (A) Representative trace of current versus time recorded from a Xenopus oocyte 24–48 h after nuclear injection of plasmid cDNA encoding mouse P2XR7. 5 μM Suramin inhibits ATP- and histone-evoked currents. (B) Average current after 30 s of antagonist treatment relative to baseline histone-evoked current (Ctrl, 30 s time/vehicle control; 5 μM PPADS, 10 μM TNP-ATP, 5 μM Suramin, Ave ± SEM, n = 3–4 animals, 7–18 cells, P values from ANOVA with multiple comparisons are reported in the figure). (C–H) Representative trace and average ATP-evoked current after application of indicated P2XR7-selective antagonists on ATP-activated, open, channels. (D) 10 μM AZ10606120 (AZ10, Ave ± SEM, n = 3 animals, 18 cells). (E) 10 μM A438079 (A43, Ave ± SEM, n = 2 animal, 8 cells). (F) 50 μM GW791343 (GW, Ave ± SEM, n = 5 animals, 16 cells). (G) 10 μM AZ11645373 (AZ11, Ave ± SEM, n = 3 animals, 8 cells). (H) 1 μM KN-62 (KN62, Ave ± SEM, n = 2 animals, 7 cells). P values by Student’s t test indicated above each graph. (I–N) Representative trace and average histone-evoked current after application of indicated P2XR7-selective antagonist on histone-activated, open, channels. (J) 10 μM AZ10606120 (AZ10, Ave ± SEM, n = 3 animals, 10 cells). (K) 10 μM A438079 (A43, Ave ± SEM, n = 2 animals, 8 cells). (L) 50 μM GW791343 (GW, Ave ± SEM, n = 2 animals, 9 cells). (M) 10 μM AZ11645373 (AZ11, Ave ± SEM, n = 3 animals, 8 cells). (N) 1 μM KN-62 (KN62, Ave ± SEM, n = 1 animal, 3 cells). P values by Student’s t test indicated above each graph. (O–Q) Representative trace (O) and average ATP- and histone-evoked currents before (control) and after pre-application of P2XR7-selective antagonists. (P) ATP-evoked currents (Ave ± SEM) after pre-application of AZ10 (n = 2 animals, 9 cells), A43 (n = 2 animals, 8 cells), GW (n = 3 animals, 9 cells), AZ11 (n = 3 animals, 9 cells), or KN62 (n = 2 animals, 7 cells). (Q) Histone-evoked currents (Ave ± SEM) after pre-application of AZ10 (n = 3 animals, 15 cells), A43 (n = 3 animals, 11 cells), GW (n = 2 animals, 9 cells), AZ11 (n = 2 animals, 7 cells), or KN62 (n = 2 animals, 9 cells). P values by one-way ANOVA (non-parametric) indicated above the graph.

Histone-evoked P2XR7 currents are increased in low Ca2+ conditions

One feature of P2XR7 is that ATP-evoked currents are increased in low extracellular Ca2+ conditions (North and Surprenant, 2000; Yan et al., 2011). Prolonged activation by ATP in low Ca2+ causes P2XR7 pore formation that allows conductance of large cations and cationic nuclear dyes (Kobayashi et al., 1989; Innocenti et al., 2004; Karasawa et al., 2017). However, with the high concentrations of ATP required to activate P2XR7, it is unclear if P2X7 activation by millimolar [ATP] is compounded by ATP chelation of Ca2+ and is, therefore, ATP-dependent. We tested the effect of extracellular Ca2+ on histone-evoked P2XR7 currents. While histone-evoked currents in 2 mM Ca2+ Ringer’s were rapidly reversed (Fig. 1 A), histone-evoked P2XR7 currents in low Ca2+ conditions (0.4 mM) were significantly larger and were only partially reversed by washing (Fig. 3, A and B). Subsequent wash with 2 mM Ca2+ Ringer’s reversed the remaining histone-evoked P2XR7 current back to baseline (Fig. 3 A). These results demonstrate that histone-evoked P2XR7 currents are also inhibited by extracellular Ca2+.

Figure 3.

Figure 3.

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Histone-evoked P2XR7 currents are increased in low Ca2+ conditions. (A) Representative trace of current versus time recorded from a Xenopus oocyte 24–48 h after nuclear injection of plasmid cDNA encoding mouse P2XR7. Histones were applied in low Ca2+ solution and washed with high Ca2+. (B) P2XR7 histone currents in high and low Ca2+ (Ave ± SEM, n = 5–9 animals, 19–26 cells, * P < 0.0001 by unpaired Student’s t test).

Mouse P2XR7 variants, 451L and -P, are activated by ATP and histones

The C57BL/6J mouse line (#000664; Jackson Labs) carries a P2XR7 variant (P451L) that is reported to have reduced function (Adriouch et al., 2002). We tested the hypothesis that the 451L variant impairs P2XR7 currents by comparing mouse P2XR7 451L (C57 WT) and 451P function in oocytes. Contrary to expectation, we found no deficit in 451L ATP-evoked currents (Fig. 4). ATP concentration response curves from 451P- and 451L-expressing cells were not significantly different (Fig. 4 A). P2XR7 451L currents, in response to 500 μM ATP, were statistically significantly larger than 451P currents (Fig. 4 B). We also tested for differences in histone-evoked 451L and 451P currents. Histone-evoked P2XR7 451L currents were not significantly different from histone-evoked 451P currents (Fig. 4 C). The data demonstrate that there is no intrinsic functional deficit in the C57 P2XR7 451L variant with regard to ATP- or histone-evoked P2XR7 currents.

Figure 4.

Figure 4.

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Mouse P2XR7 variants, 451L and -P, are activated by ATP and histones. (A) Average ATP-evoked current as a function of ATP concentration from cells expressing mouse P2XR7 451P and 451L variants (Ave ± SEM, n = 4–7 animals, 13–21 cells). (B) Average ATP-evoked (500 μM) currents for 451P and 451L variants (Ave ± SEM, n = 4–7 animals, 20–21 cells, P = 0.0482). (C) Average histone-evoked (20 μg/ml) currents for 451P and 451L variants (Ave ± SEM, n = 4–7 animals, 20–21 cells, P = 0.2709).

Histone H2b, H3, and H4, but not H2a, activate P2XR7 currents

In prior work, we found that purified, unfractionated, histone proteins cause EC Ca2+ influx in human and mouse resistance-size mesenteric arteries (Collier et al., 2019). In this series of experiments, we demonstrate that these same core histone proteins activate P2XR7 (Figs. 1, 2, 3, and 4). Using the same experimental conditions, we tested the effect of individual, recombinant, core histone proteins (H2a, H2b, H3, and H4) on P2XR7 currents. As shown in Fig. 5, H2b, H3, and H4, but not H2a, were individually capable of activating P2XR7 currents. Our findings demonstrated that the current properties differed between each of the tested histone subunits (Fig. 5, A and B). More specifically, H2b most closely resembled currents activated by unfractionated histones (Fig. 5 A). H2b-evoked currents did not desensitize, but current returned to baseline after wash (Fig. 5 A). Histones H3 and H4 activate rapidly desensitizing P2XR7 currents (Fig. 5 B). These results demonstrate the specificity of P2XR7 histone-evoked currents, confirm that histone proteins are sufficient to activate P2XR7 current, and suggest that each histone protein subunit may have a unique site and mechanism of P2XR7 current activation.

Figure 5.

Figure 5.

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Histone H2b, H3, and H4, but not H2a, activate P2XR7 currents. (A and B) Representative trace of P2XR7 currents at −60 mV activated by purified recombinant human H2b (A), H3 (B, top), and H4 (B, bottom; 1 μg/ml, each). (C) Average histone-evoked current at −60 mV in control (Ctrl) and mouse P2XR7 (451L) expressing oocytes (n = 2–3 animals, 6–17 cells, P = 0.291 (H2a), P = 0.0002 (H2b), P = < 0.0001 (H3), P < 0.0001 (H4) by unpaired Student’s t test (P2XR7 expressing versus control).

Discussion

It has been shown that the detrimental effects of circulating histones can be mitigated by histone-directed antibodies; however, many questions remain about the mechanism of extracellular histone-mediated pathology (Xu et al., 2009). Herein, we provide significant evidence that histone proteins activate P2XR7 channel currents when expressed in heterologous cells. Heterologous expression enables confirmation of P2XR7 function without having to rely on poorly selective P2XR agonists and antagonists to interpret results. Using this system, we have shown that P2XR7 is necessary and sufficient for histone-evoked currents in a non-mammalian expression system. Histone-evoked P2XR7 currents exhibit many of the same properties as ATP-evoked P2XR7 currents. Histone-evoked P2XR7 currents reverse at the same potential as ATP-evoked P2XR7 currents, are not voltage-sensitive, and are potentiated in low Ca2+ conditions. Additionally, histone-evoked P2XR7 currents are dependent on intact histone proteins and demonstrate histone protein subunit-specific differences in activation and desensitization kinetics. Based on the kinetics of current decay alone, histone proteins may be a more potent agonist of P2XR7 than ATP or BzATP. This is physiologically significant as the high levels of ATP needed to activate P2XR7 may be more difficult to achieve in circulation than the levels of histone proteins needed to activate P2XR7. We demonstrate that P2XR7 is activated by histone concentrations at the low end of the range reported in circulation after traumatic injury or in ARDS (Fig. 1; Abrams et al., 2013; Lv et al., 2017). Thus, in some cases, extracellular histone proteins may be a more relevant agonist of P2XR7 than extracellular ATP.

One hypothesis reviewed in the literature regarding the mechanism of action for circulating histone proteins describes a receptor-independent mechanism where histone protein subunits directly interact with the plasma membrane and form a non-selective pore (Silk et al., 2017). This was not observed in our experiments. If the mechanism of extracellular histone proteins is receptor-independent, application of histone proteins would be expected to cause currents in control (non-P2XR7 expressing) cells and create a non-selective leak current that would reverse near 0 mV and/or current might be voltage-dependent if basic histone residues are crossing or fully inserting into the membrane. We only observed histone-evoked currents in cells expressing P2XR7. In P2XR7 expressing cells, ATP- and histone-evoked reversal potentials are not statistically different, suggesting that histone exposure does not create a new, P2XR7-independent, conduction pathway. If histone proteins were fully inserting into the plasma membrane or interacting with P2XR7 membrane spanning domain, basic residues would be exposed to the electric field of the cell and we would expect the response to be voltage-sensitive. Our results demonstrated that histone-evoked currents were not voltage-sensitive. Therefore, we speculate that histone proteins may be allosterically regulating P2XR7 function through an extracellular domain interaction. However, this evidence should not be interpreted as sufficient to exclude the possibility of membrane or P2XR7 transmembrane domain interaction as histone proteins could be interacting in a conformation in which basic residues remain in the extracellular space and are not exposed to the electric field across the membrane. Future structural and functional studies directed at identifying the histone-binding residues are necessary to definitively determine the P2XR7 histone-binding region.

Prior work on histone-induced EC Ca2+ influx was performed on resistance-size mesenteric arteries from human tissue or C57BL/6J background mice (Collier et al., 2019). The C57BL/6J mouse line (#000664; Jackson Labs) carries a P2XR7 variant (P451L in the 595 aa isoform) that is reported in Mouse Genome Informatics as an allele with “severely reduced activity.” This conclusion was based on correlations between mouse genetic backgrounds without direct P2XR7 current records (Adriouch et al., 2002). If this is an intrinsic property of the channel, we would anticipate decreased P2XR7 451L function when expressed in a heterologous system. We directly tested the hypothesis that the 451L variant impairs P2XR7 currents by comparing mouse P2XR7 451L (C57 WT) and 451P function in oocytes. We found no deficit in P2XR7 451L in ATP- or histone-evoked currents relative to 451P.

Previous work examined the cytotoxicity of unfractionated histone proteins (at 50 μg/ml rather than 10 μg/ml used herein) and individual purified histone subunits (H2a, H2b, H3, and H4; 20 μg/ml each versus 1 μg/ml herein) on HUVECs using a PI uptake assay (Xu et al., 2009). While it is not possible to know if PI uptake in this assay was indicative of cell death or P2XR7 activation, there appear to be some overlap in findings. Interestingly, H2a showed lower PI uptake than unfractionated histones or H3 and H4 (Xu et al., 2009). In our work, consistent with these previous findings, we find that H2b, H3, and H4 appear to be the most potent P2XR7 activators. While H2b alone most closely recapitulates activation by unfractionated histones, H3 and H4 histone-evoked currents are reminiscent of rapidly desensitizing ATP-evoked currents typically observed with other P2XR family members (reviewed in North, 2002). Additionally, Xu et al. (2009) demonstrated protective effects of anti-H4 antibodies in mice challenged with LPS. While this is consistent with our data on the activity of H4, the tissue level localization of P2XR7 pathways potentially impacted are unclear with systemic delivery of anti-histone antibody. Histone proteins are also subject to posttranslational modifications, such as citrullination, that have been reported to be elevated in disease (Paues Goranson et al., 2018). It will be important to continue to pursue these differences to understand physiologic consequences of activation by different histone proteins and modifications present in circulation.

Our finding that P2XR7-selective antagonists fail to inhibit histone-evoked P2XR7 currents demonstrates the advantages of recording ion channel currents in a heterologous expression system. In native P2XR7 tissue, if P2XR7-selective antagonists fail to inhibit histone-evoked current or dye uptake, it would be logical to conclude that P2XR7 is not directly involved. However, as we demonstrate, histone proteins clearly activate P2XR7 currents despite not being blocked by P2XR7-selective antagonists. A similar effect has been reported with LL-37 stimulated dye uptake. LL-37 increases dye uptake in P2XR7 expressing cells (Tomasinsig et al., 2008). However, P2XR7-selective antagonists that inhibit BzATP-stimulated dye uptake in P2XR7 expressing HEK293, cells do not inhibit LL-37–stimulated dye uptake (Jackson et al., 2022). One disadvantage of dye uptake assays is that increases in nuclear dye accumulation could be the result of recruitment of other channels or cell death. Ion channel current records provide a more specific, definitive, assay of P2XR7 function. Here, we demonstrated that histone proteins only evoke currents in P2XR7 expressing cells, with properties that resemble ATP-evoked P2XR7 currents. The inability of P2XR7-selective antagonists to inhibit histone-evoked currents and ATP-stimulated dye uptake may be due to differences in allosteric modulation of channel activity. New structural data will be needed in the future to address this question and will hopefully lead to more comprehensive pharmacology.

Our results demonstrated that histone proteins activate P2XR7 channel currents. Given the known importance of P2XRs and circulating histone proteins in immunity, vascular function, pain sensation, and bone formation; many avenues are available for future work exploring the role of histone-P2XR signaling and development of new pharmacologic and therapeutic strategies.

Acknowledgments

Jeanne M. Nerbonne served as editor.

We thank Kasey D. Stewart, Jordan Simien, and Ariel Lane for technical assistance.

D.M. Collier is supported by the National Institutes of Health (grant R00HL133451).

Author contributions: R. Al-Aqtash: conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing—review & editing; M.S. Ross: data curation, formal analysis, investigation, writing—review & editing; D.M. Collier.: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization, writing—original draft, writing—review & editing.

Data availability

Original data are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Original data are available from the corresponding author upon reasonable request.

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

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