Functional Coupling between the P2X7 Receptor and Pannexin-1 Channel in Rat Trigeminal Ganglion Neurons

The ionotropic P2X receptor, P2X7, is believed to regulate and/or generate nociceptive pain, and pain in several neuropathological diseases. Although there is a known relationship between P2X7 receptor activity and pain sensing, its detailed functional properties in trigeminal ganglion (TG) neurons remains unclear. We examined the electrophysiological and pharmacological characteristics of the P2X7 receptor and its functional coupling with other P2X receptors and pannexin-1 (PANX1) channels in primary cultured rat TG neurons, using whole-cell patch-clamp recordings. Application of ATP and Bz-ATP induced long-lasting biphasic inward currents that were more sensitive to extracellular Bz-ATP than ATP, indicating that the current was carried by P2X7 receptors. While the biphasic current densities of the first and second components were increased by Bz-ATP in a concentration dependent manner; current duration was only affected in the second component. These currents were significantly inhibited by P2X7 receptor antagonists, while only the second component was inhibited by P2X1, 3, and 4 receptor antagonists, PANX1 channel inhibitors, and extracellular ATPase. Taken together, our data suggests that autocrine or paracrine signaling via the P2X7-PANX1-P2X receptor/channel complex may play important roles in several pain sensing pathways via long-lasting neuronal activity driven by extracellular high-concentration ATP following tissue damage in the orofacial area.

Therefore, this study was designed to examine the electrophysiological and pharmacological characteristics of the P2X 7 receptor and its functional coupling with the PANX1 channel as well as other P2X receptors in TG neurons.

Cell Identification of TG Neurons
TG neurons were identified as cells that expressed voltage-dependent ionic currents. The voltage-dependent inward currents (I inward ) were recorded at a holding potential (Vh) of −60 mV, and neurons were identified as cells showing inward currents ( Figure 1A). The current-voltage (I-V) relationships, which were determined by plotting the peak current amplitudes in the densities against the applied membrane potentials, were measured by applying 20-ms voltage steps ranging from −80 to +100 mV at 2-s intervals ( Figure 1A,B). At a Vh of −60 mV, the inward currents in the neurons developed at a membrane potential of −30 mV and reached maximum amplitude at a membrane potential of −10 mV ( Figure 1B). Cells not showing inward currents were not taken into account in further analyses.

Response of TG Neurons during Application of ATP and Bz-ATP
We examined the concentration-dependent effects of both ATP and Bz-ATP on the induced-current response in TG neurons. In the standard ECS, application of 100 µM Bz-ATP (a highly potent P2X 7 receptor agonist), as well as 100 µM or 10 mM, of ATP evoked biphasic inward currents at Vh of −60 mV (Figure 2A), composing the transient first component and the persistent second component, respectively. The currents were elicited by four different concentrations of Bz-ATP (1-100 µM) and ATP (100 µM-10 mM). The amplitudes of current densities increased with increasing Bz-ATP and ATP concentrations in a concentration-dependent manner ( Figure 2B). The EC 50 of ATP on its induced-currents was 3.72 mM, whereas the EC 50 of Bz-ATP on its induced-currents was 28.5 µM. This is a 160-fold difference in the EC 50 values of Bz-ATP and ATP. of −60 mV, and neurons were identified as cells showing inward currents ( Figure 1A). The current-voltage (I-V) relationships, which were determined by plotting the peak current amplitudes in the densities against the applied membrane potentials, were measured by applying 20-ms voltage steps ranging from −80 to +100 mV at 2-s intervals ( Figure 1A,B). At a Vh of −60 mV, the inward currents in the neurons developed at a membrane potential of −30 mV and reached maximum amplitude at a membrane potential of −10 mV ( Figure  1B). Cells not showing inward currents were not taken into account in further analyses.

Response of TG Neurons during Application of ATP and Bz-ATP
We examined the concentration-dependent effects of both ATP and Bz-ATP on the induced-current response in TG neurons. In the standard ECS, application of 100 µM Bz-ATP (a highly potent P2X7 receptor agonist), as well as 100 µM or 10 mM, of ATP evoked biphasic inward currents at Vh of −60 mV (Figure 2A), composing the transient first component and the persistent second component, respectively. The currents were elicited by four different concentrations of Bz-ATP (1-100 µM) and ATP (100 µM-10 mM). The amplitudes of current densities increased with increasing Bz-ATP and ATP concentrations in a concentration-dependent manner ( Figure 2B). The EC50 of ATP on its induced-currents was 3.72 mM, whereas the EC50 of Bz-ATP on its induced-currents was 28.5 µM. This is a 160-fold difference in the EC50 values of Bz-ATP and ATP.

Concentration-Dependent Effect of Bz-ATP on the Biphasic Inward Current
To investigate the detailed concentration-dependent effect on the current response, we examined four different concentrations of Bz-ATP (1-100 µM). Biphasic inward currents were elicited by different concentrations of Bz-ATP ( Figure 3A). The current densities in both the first and second components of the Bz-ATP-induced inward currents increased in a concentration-dependent manner ( Figure 3B). The EC 50 obtained in the first and second components were 28.5 µM and 29.4 µM ( Figure 3B), respectively. Additionally, the duration of the second component increased in a concentration-dependent manner (EC 50 of 18.2 µM of Bz-ATP), while that of the first component remained consistent (EC 50 was not calculated; Figure 3C). differences between data points are indicated by asterisks. * p < 0.05 vs. 1 µM Bz-ATP; ** p < 0.05 vs. 100 µM ATP.

Concentration-Dependent Effect of Bz-ATP on the Biphasic inward Current
To investigate the detailed concentration-dependent effect on the current response, we examined four different concentrations of Bz-ATP (1-100 µM). Biphasic inward currents were elicited by different concentrations of Bz-ATP ( Figure 3A). The current densities in both the first and second components of the Bz-ATP-induced inward currents increased in a concentration-dependent manner ( Figure 3B). The EC50 obtained in the first and second components were 28.5 µM and 29.4 µM ( Figure 3B), respectively. Additionally, the duration of the second component increased in a concentration-dependent manner (EC50 of 18.2 µM of Bz-ATP), while that of the first component remained consistent (EC50 was not calculated; Figure 3C).  Figure 2B. Each data point represents the mean ± SD of five separate experiments. Statistically significant differences between data points are indicated by asterisks. * p < 0.05.

Effects of P2X Receptor Antagonists on the Bz-ATP-Induced Currents
To investigate which P2X receptors contributed to the biphasic inward current induced by Bz-ATP, we examined the effects of the non-specific P2X receptor antagonist (PPADS), the P2X1 receptor antagonist (NF449), the P2X3 receptor antagonist (NF110), the P2X4 receptor antagonists (5-BDBD and PSB-12062, respectively), and P2X7 receptor antagonists (A-740003 and A-438079, respectively). In the first component, the inward currents induced by 100 µM Bz-ATP were significantly inhibited by 50 µM PPADS, 10 µM A-740003, and 100 µM A-438079 ( Figure 4A,B). The second component was significantly  Figure 2B. Each data point represents the mean ± SD of five separate experiments. Statistically significant differences between data points are indicated by asterisks. * p < 0.05.

P2X7-PANX1-P2X Receptor/Channel Axis Mediates Biphasic inward Current Activatio Autocrine Signaling
To investigate the nature of the second component in the biphasic inward cur we examined the effect of extracellular ATP degradation enzymes and PANX1 ant nists on the current induced by Bz-ATP ( Figure 5A). Although there was no signifi effect on the first component of the biphasic inward currents induced by 100 µM Bz-  To investigate the nature of the second component in the biphasic inward current, we examined the effect of extracellular ATP degradation enzymes and PANX1 antagonists on the current induced by Bz-ATP ( Figure 5A). Although there was no significant effect on the first component of the biphasic inward currents induced by 100 µM Bz-ATP ( Figure 5B), the current densities of the second components were significantly reduced by 10 µM mefloquine (non-specific PANX1 antagonist) and 50 µM 10 Panx (specific PANX1 antagonist), as well as 100 U/mL apyrases (extracellular ATP degradation enzyme) ( Figure 5C).  Figure 5B), the current densities of the second components were significantly reduced by 10 µM mefloquine (non-specific PANX1 antagonist) and 50 µM 10 Panx (specific PANX1 antagonist), as well as 100 U/mL apyrases (extracellular ATP degradation enzyme) ( Figure 5C).

Discussion
This study revealed the functional expression and synergistic effects of P2X7, PANX1 channels, and other P2X receptors in rat TG neurons. We found that the P2X7-PANX1-P2X receptor/channel axis mediates the Bz-ATP-induced long-lasting biphasic inward current response in an autocrine manner. P2X7 receptors are activated by extracellular ATP (at a high concentration) and Bz-ATP, and are found in numerous organs [2,16,17,25,31,34], with the exception of TG neurons [22]. P2X7 receptors require activation at ATP concentrations greater than 100 µM, while Bz-ATP is approximately 10-30 times more potent than ATP [2]. In line with these observations, we found that the EC50 required to activate an inward current in TG neurons was 20 µM for Bz-ATP, but 3300 µM for ATP ( Figure 2). In addition, the first component of the biphasic inward currents induced by 100 µM Bz-ATP was reduced by P2X7 receptor antagonists, A-438079 and A-740003 ( Figure 4). These results indicate that P2X7 receptors in TG neurons are likely less sensitive to extracellular ATP but are highly sensitive to extracellular Bz-ATP. Furthermore, Bz-ATP also triggers the activation of biphasic inward currents.
We also found that application of Bz-ATP and high concentrations of ATP to TG neurons elicited an additional current component, that is, the second component, of the biphasic inward currents in a concentration-dependent manner ( Figure 3). Importantly, the

Discussion
This study revealed the functional expression and synergistic effects of P2X 7 , PANX1 channels, and other P2X receptors in rat TG neurons. We found that the P2X 7 -PANX1-P2X receptor/channel axis mediates the Bz-ATP-induced long-lasting biphasic inward current response in an autocrine manner. P2X 7 receptors are activated by extracellular ATP (at a high concentration) and Bz-ATP, and are found in numerous organs [2,16,17,25,31,34], with the exception of TG neurons [22]. P2X 7 receptors require activation at ATP concentrations greater than 100 µM, while Bz-ATP is approximately 10-30 times more potent than ATP [2]. In line with these observations, we found that the EC 50 required to activate an inward current in TG neurons was 20 µM for Bz-ATP, but 3300 µM for ATP ( Figure 2). In addition, the first component of the biphasic inward currents induced by 100 µM Bz-ATP was reduced by P2X 7 receptor antagonists, A-438079 and A-740003 ( Figure 4). These results indicate that P2X 7 receptors in TG neurons are likely less sensitive to extracellular ATP but are highly sensitive to extracellular Bz-ATP. Furthermore, Bz-ATP also triggers the activation of biphasic inward currents.
We also found that application of Bz-ATP and high concentrations of ATP to TG neurons elicited an additional current component, that is, the second component, of the biphasic inward currents in a concentration-dependent manner (Figure 3). Importantly, the second component of biphasic inward currents induced by Bz-ATP was affected by other P2X receptor antagonists (predominantly the P2X 4 antagonist, but also the P2X 1 and P2X 3 antagonists) (Figure 4) as well as the PANX1 channel inhibitor and extracellular ATPase ( Figure 5), although the first component did not show any sensitivity to these antagonists ( Figure 4). It has been reported that P2X receptor-induced activation of the PANX1 channel is a relatively late event, which is preceded by the rapid opening of the ionotropic receptor, and occurs only after prolonged agonist application [35]. The PANX1 channel assists with autocrine and/or paracrine signaling by providing an extracellular signaling pathway via ATP [36][37][38]. Moreover, it has been reported that functional interactions between the P2X 7 and/or P2X 4 receptor and PANX1 channel form a complex that interact directly with each other [30,39]. Together with these reports, our results suggest that activation of the P2X 7 receptor most likely enhances PANX1 channel opening enabling the extracellular release of ATP which activates other P2X receptors, especially the P2X 4 receptor, resulting in the generation of biphasic inward currents through an autocrine mechanism.
The P2X 7 -PANX1-P2X receptor/channel has important roles in both physiological and pathological conditions [35,40,41]. In the inflammatory responses, the interaction between P2X 7 receptors and PANX1 channels promotes cytosolic multiprotein oligomerization that leads to the activation of the inflammasome, followed by the extracellular release of cytokines [42]. In contrast, besides inducing inflammasome, ATP also influences the activation and chemoattraction of leukocytes to the injury site to amplify the immune response. Multinucleated macrophage formation takes place following ATP release through P2X 7 receptors and subsequent conversion into adenosine to enhance the expression of CD44, while PANX1 allows the permeabilization of the plasma membrane, thereby enabling fusion [42]. These inflammatory-and/or immuno-responses lead to inflammation-induced cell death, that is pyroptosis. In pyroptosis, released ATP via PANX1 channels activates P2X receptors, which underlies the formation of a membrane pore that triggers cytolysis [42]. It has also been reported that the activation of the neuronal P2X 7 -PANX1 axis mediates the death of enteric neurons during colitis [25], while the activation of the P2X 7 -PANX1 complex enhances spreading depolarization and neuroinflammation [9] of the trigeminovascular system, leading to migraine and stroke. Therefore, there is considerable interest in understanding whether the mechanisms of pyroptosis in the peripheral trigeminal nervous system are involved in the generation and/or modulation of neuropathic pain (represented as postherpetic neuralgia, trigeminal neuralgia, and postoperative neuropathic pain) and/or trigeminal autonomic cephalalgias. This study was conducted to determine the electrophysiological and pharmacological characteristics of the P2X 7 receptor and its functional coupling mediated by the P2X 7 -PANX1-P2X receptor/channel complex in trigeminal ganglion neurons. Thus, to reveal the detailed role of the P2X 7 -PANX1-P2X axis in the generation of pain under both physiological and pathophysiological conditions, further studies, such as analyzing pain behaviors induced by stimuli in the orofacial area (personal communication from YS), are of immediate interest.
This study has several limitations. First, the first component of the Bz-ATP-induced biphasic current was not completely suppressed by the P2X 7 receptor-selective antagonists, A-438079 and A-740003. Therefore, a residual current component could still be observed (Figure 4), suggesting that the first component of the current may not be completely mediated by the P2X 7 receptor. In fact, Bz-ATP has been reported to act on various recombinant homomeric P2X receptors (i.e., P2X 1 , P2X 2 , P2X 3 , P2X 4 , P2X 5 , and P2X 7 ), and the presence of heteromeric P2X 4/7 receptors has been proposed [43]. Furthermore, P2X 1 -P2X 6 receptor subtypes have been reported in rat TG neurons [1,7,8]. Moreover, large pore openings giving a biphasic current can also be generated by P2X 2 and P2X 4 receptors [44]. Finally, P2X 2 receptors are also expressed in TG neurons [45]. In this study, however, the first component of the Bz-ATP-induced current did not show sensitivity to P2X 1 , P2X 3 , and P2X 4 receptor-selective antagonists. PPADS, a non-selective P2X receptor antagonist, blocks recombinant P2X 1 , P2X 2 , P2X 3 , and P2X 5 receptors (IC 50 = 1-3 µM), and is effective against P2X 7 at high concentrations (IC 50 = 50 µM) [43]. Based on the present results showing that the first component could not be inhibited by low concentrations (10 µM) of PPADS but was inhibited by high concentrations (50 µM), the contribution of P2X 2 and P2X 5 receptor activation to the first component of the Bz-ATP-induced inward current is unlikely. Thus, we suggest that the activation of the first component is predominantly P2X 7 receptor-dependent, and further studies are needed to clarify the contribution of P2X 2 and P2X 5 receptors to the first component of the Bz-ATP-induced biphasic current.
Since selective antagonists for P2X 2 and P2X 5 receptors are not commercially available, detailed contributions of these P2X subtypes could not be pursued in this study. The concentration of P2X 7 receptor antagonists used in this study was 10-100 µM, while the reported IC 50 for these reagents is approximately 50 nM [43]. This is because the currents were not sufficiently inhibited until the concentration was raised up to the µM order in rat TG neurons. Importantly, the IC 50 of some P2X receptor antagonists has also been reported to be highly species-dependent. The amino acid residue at position 95 of the P2X 7 receptor is key and determines species-dependent selectivity of allosteric antagonists (such as GW791343, KN62, and SB203580). The P2X 7 receptor antagonists, A740003 and A438079, which we used in this study, also show allosteric effects [46]. Therefore, the species-dependent selectivity of the allosteric antagonists might be the reason as to why the 10-100 µM order of P2X 7 receptor antagonist concentration was necessary to inhibit the Bz-ATP-induced biphasic currents in this study. Examples using 30 µM A438079 to inhibit P2X 7 receptor activity have also been recently reported [25]. In addition, in line with our present results (see above too), this residue (at position 95) does not affect the species-dependent selectivity of PPADS, which is a slowly reversible antagonist of the P2X 7 receptor and acts at the ATP-binding site [47].
Second, we showed that extracellular ATP released from the PANX1 channel can activate the P2X 1 , P2X 3 , and P2X 4 receptors in the same TG neuron as an autocrine signal that is triggered by P2X 7 receptor activation. However, we were unable to estimate the amount and extracellular concentration of ATP released from the PANX1 channel, as well as its diffusible constant. The basal level of extracellular ATP is low, in nanomolar levels, in the trigeminal ganglion neurons to avoid constant nociceptive firing in trigeminal nerves [26]. In contrast, activation of P2 receptors usually occurs at nucleotide concentrations within the range of 10 −6 -10 −3 M, which exceeds the nanomolar ATP level at rest ( [48] for review). A recent innovative ATP-sensing assay revealed that 100-200 µM ATP is released in response to P2X 7 receptor activation by Bz-ATP [49]. Although further studies are needed to estimate the amount of ATP released following the activation of the P2X 7 -PANX1 axis, up to µM order of ATP release might be reasonable to activate P2 receptors expressed in TG neurons.
In conclusion, our study suggests that the functional P2X 7 -PANX1-P2X complex elicits long-lasting biphasic inward currents in TG neurons. This autocrine and/or paracrine signaling via ATP may contribute to the pain mechanism in the orofacial region.

Th Cell Isolation and Primary Culture
All animals were treated in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences approved by the Council of the Physiological Society of Japan and by the American Physiological Society. This study also followed the guidelines established by the National Institutes of Health (Bethesda, MD, USA) regarding the care and use of animals for experimental procedures. The study was approved by the Ethics Committee of Tokyo Dental College (approval No.282804, 292502, 302502, 192701).

Whole-Cell Recording Techniques
Patch-clamp recordings of the whole-cell configuration were performed under voltageclamp conditions. Patch pipettes with a resistance of 2-5 MΩ were pulled from capillary tubes using a DMZ-Universal Puller (Zeitz Instruments, Martinsried, Germany), and the pipettes were filled with an intracellular solution (ICS; solution composition is described below). Whole-cell currents were measured using a patch-clamp amplifier (L/M-EPC-7+; Heka Electronik, Lambrecht, Germany). Current traces were displayed and stored in a computer using pCLAMP (Molecular Devices, LLC., San Jose, CA, USA) after digitizing the analog signals at 10 kHz (DigiData 1440A; Molecular Device). The current records were filtered at 3 kHz. Data were analyzed offline using pCLAMP. All experiments were performed at 25 • C.

Solutions and Reagents
HBSS was used as the standard extracellular solution (ECS) for patch-clamp recordings. The intracellular solution (ICS) for patch-clamp recordings contained the following: 140 mM KCl, 10 mM NaCl, and 10 mM HEPES (pH was adjusted to 7.2, using Tris). The following pharmacological agents were used: ATP disodium salt (Sigma-Aldrich Co.) and Bz-ATP (Sigma-Aldrich Co.) as P2X receptor agonist, 3- ) and Trp-Arg-Gln-Aln-Phe-Val-Asp-Ser-Tyr ( 10 Panx) (Tocris Bioscience, Bristol, UK) as PANX1 channel inhibitors, as well as ATP diphosphohydrolase adenosine 5 -triphosphatase (apyrase) (Sigma-Aldrich Co.). These reagents were diluted in ECSs for patch-clamp recording and applied at the appropriate concentration to primary cultured TG neurons via a rapid solution exchange system (Warner Instruments, Hamden, CT, USA).

Analyses
Differences in cell size were accounted for by normalizing the measured capacitance. In this study, the induced current amplitudes are expressed in terms of the current densities (pA/pF). Data are expressed as the mean ± standard deviation (S.D.) of n observations, where n represents the number of separate experiments. The concentration dependence of Bz-ATP activity was obtained by fitting the data with the following function: where EC 50 is the half-maximal effective concentration of applied Bz-ATP, A max is the maximal response, and A min is the minimal response.
[X] o indicates the concentration of extracellular Bz-ATP. One-way ANOVA with Bonferroni correction was used to determine parametric statistical significance; the Kruskal-Wallis test with Dunn's posthoc test was used to determine nonparametric statistical significance. All statistical analyses were performed using Graph-Pad Prism version 7 statistical software package (GraphPad Software, San Diego, CA, USA).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.