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. Author manuscript; available in PMC: 2021 Jul 15.
Published in final edited form as: Neuroscience. 2019 Jul 23;439:332–341. doi: 10.1016/j.neuroscience.2019.07.025

Tonic calcium-activated chloride current sustained by ATP release and highly desensitizing human P2X1 receptors

Agenor Limon 1,2,*, Garo Hagopian 2, Jorge M Reyes-Ruiz 2, Ricardo Miledi 2,†
PMCID: PMC6980929  NIHMSID: NIHMS1535527  PMID: 31349005

Abstract

Extracellular adenosine triphosphate (ATP) participates in maintaining the vascular tone in the CNS, particularly in the retina, via the tonic activity of ligand gated activated P2X1 receptors. P2X1 receptors are characterized by their high affinity for ATP and their strong desensitization to concentrations of ATP that are 200-fold lower than their EC50. The mechanism behind P2X1 tonic activity remains unclear. In this study, we expressed human P2X1 (hP2X1) homomeric receptors in Xenopus oocytes to explore the relationship between ATP release from oocytes at rest, hP2X1, and Ca2+-activated Cl− channels. Our results indicate that Xenopus oocytes release ATP at rest via vesicular exocytosis, and this process is a constitutive phenomenon independent of extracellular Ca2+. Our results also indicate that hP2X1 receptors are able to sustain a tonic activity of Ca2+-activated Cl− channels. In the presence of extracellular Ca2+ the activity of hP2X1 receptors is greatly amplified by its coupling with Ca2+-activated Cl− channels. Future studies addressing the relationship between hP2X1 receptors and Ca2+-activated Cl− channels in vascular smooth muscle cells should provide information about additional mechanisms that regulate the vascular tone and their potential as pharmaceutical targets.

Keywords: ATP release, vascular tone, purinergic signaling, tonic activity

Introduction

Adenosine triphosphate (ATP) is one of the most versatile molecules in living systems with a number of roles in intracellular and extracellular signaling (Burnstock, 2014). Most cells, if not all, release ATP to the extracellular medium by vesicular exocytosis, ATP-permeable channels, or a combination of both (Lazarowski, 2012; Praetorius and Leipziger, 2009). Therefore, the paracrine and autocrine effects of ATP depend on the cell membrane receptors and the intracellular signaling downstream of their activation (Fields and Stevens, 2000; Kur and Newman, 2014). Extracellular signaling triggered by ATP can be mediated by G-coupled P2Y receptors or ionotropic P2X receptors (Burnstock, 2014). Of the seven subunits (P2X1-7) in the P2X family, P2X1 and P2X3 are characterized by their high affinity and strong desensitization to ATP, even at concentrations near 200-fold lower than their EC50 (McDonald et al., 2002; Rettinger and Schmalzing, 2003; Rettinger and Schmalzing, 2004). Nonetheless, P2x1 receptors in vascular smooth muscle cells have been shown to participate in sustaining the vascular tone of retina vessels (Kur and Newman, 2014), suggesting that P2X1 are able to be continuously open despite their strong desensitization. The mechanism behind their tonic activity is unclear.

Vascular smooth muscle cells express Ca2+-activated Cl− channels (CaCCs) which are important in vascular contractility and the regulation of blood pressure (Heinze et al., 2014). These channels release ATP during mechanical stimulation produced by shear stress (Lohman et al., 2012). Similar characteristics have been observed in Xenopus oocytes. Oocytes express endogenous CaCCs (Miledi, 1982) and release ATP at rest and during mechanical stimulation (Aleu et al., 2003; Bahima et al., 2006; Bodas et al., 2000; Maroto and Hamill, 2001; Saldana et al., 2009; Saldana et al., 2005). Homomeric P2X1 receptors have been characterized in Xenopus oocytes, but due to their Ca2+ permeability, their study has usually been done in conditions that reduce the participation of CaCCs (e.g. using Ba2+ instead of Ca2+) (Valera et al., 1994). In this study, we examined heterologously expressed human P2X1 (hP2X1) in Xenopus oocytes in order to explore the relationship between ATP release from oocytes, hP2X1, and Ca2+ activated Cl− channels.

Experimental Procedures

Heterologous expression of hP2X1 receptors

Human P2X1 (hP2X1) cDNA was purchased from OriGene (Rockville, MD). The plasmid was transformed into E. coli Top 10 strain for storage and amplification. Linearized plasmids were used as templates for cRNA synthesis using the Ambion’s mMessage mMachine kit (Ambion; Austin, TX). Fifteen nl of cDNA or 50 nl cRNA (concentration 0.5 to 1 mg/ml) were injected into the nucleus or the cytoplasm of stage V–VIXenopus oocytes. Injected oocytes were kept in Barth’s solution [88 mM NaCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 1 mM KC1, 0.82 mM MgSO4, 2.4 mM NaHCO3, 10 mM Hepes (pH 7.4)] added with penicillin 100 U/ml and streptomycin 0.1 mg/ml (Sigma; St Louis, MO) at 16–17°C until the moment of recording.

Electrophysiological assay and data analysis

One to four days after injection, oocytes were impaled with two microelectrodes filled with 3 M KC1 and voltage clamped at −80 mV using a two-electrode voltage-clamp amplifier; GeneClamp 500 (Axon Instruments). Oocytes were continuously perfused with gravity-driven frog Ringer’s solution [115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 5 mM Hepes (pH 7.4)] at room temperature (19–21°C). In some experiments we used Ba2+ to replace Ca2+ (Valera et al., 1994) to reduce the participation of endogenous CaCCs (Miledi, 1982). Ba2+ permeates hP2x1 receptors but has lower apparent affinity for CaCCs compared to Ca2+. (0.46 μM vs 134 μM; (Ni et al., 2014)). To block the effect of intracellular Ca2+ oocytes were injected with 0.5 M EGTA (pH 7 with KOH) as described before (Arellano and Miledi, 1993; Miledi and Parker, 1984), using a PV820 pneumatic picopump (World Precision Instruments, Sarasota, FL) with a 10 psi pulse of 0.5 s duration. Additionally, oocytes were incubated in 10 or 50 μM 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetra acetic acid-AM (BAPTA-AM) overnight. The final concentration of DMSO in working solution was never above 0.1% as previously reported elsewhere (Saldana et al., 2009). To test whether Golgi transport participated in the generation of the STICs oocytes were incubated in 20 μM brefeldin in Barth’s solution for at least 3 h. All drugs were from sigma, NF023 was from Sigma and from Tocris. Data acquisition was performed using WinWCP V 3.9.4 at 1 KHz sampling interval (John Dempster, Glasgow, United Kingdom) as previously reported (Limon et al., 2016). Electrophysiological recordings were analyzed off line using Clampfit 10 suit software (Molecular devices; San Jose, CA). Experimental data are shown as mean ± SEM, unless otherwise stated. Statistical differences between two groups that passed the equal variance test were determined by Student’s t-test and considered significant when P < 0.05, using JMP 14 (SAS Institute; Cary, NC). For two groups with unequal variances we used the Mann-Whitney U statistic with Sigma Plot 11 (Systat, San Jose, CA). For multiple groups we used Oneway ANOVA, followed by post-hoc multiple comparisons Dunnett’s method vs control. Pearson product moment correlations were done with JMP 14.

Results

Spontaneous vesicular release of ATP

Mechanical deformation of defolliculated oocytes expressing hP2X1, by stopping (off) or reinitiating (on) the perfusion flow (Fig. 1A), elicited inward currents with amplitudes that correlated with the size of the current generated by 100 μM ATP (r = 0.86, p = 0.002, (n = 9), Pearson’s product-moment correlation; data not shown), confirming previous work demonstrating that mechanical stimulation of the oocyte induces a large release of vesicular ATP (Maroto and Hamill, 2001; Saldana et al., 2009). Interestingly, oocytes expressing hP2X1 showed spontaneous transient inward currents (STICs) that began in parallel to the current elicited by ATP (Fig. 1B). Perfusion of 100 μM ATP elicited a fast-inward current followed by a long-lasting and strong desensitization of hP2X1 receptors that is free from any STICs. This suggests that STICs result of the activation of homomeric hP2X1 receptors expressed in the oocyte membrane. Interestingly, some oocytes with STICs did not show off/on mechanical responses, suggesting that mechanical stimulations and STICs are dissociable phenomena.

Fig. 1.

Fig. 1.

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Spontaneous transient inward currents (STICs) in Xenopus oocytes expressing hP2X1 receptors. A. Oocytes expressing hP2X1 receptors, voltage-clamped at −80 mV, showed inward currents during mechanical deformation induced by stopping (off) or reinitiating (on) the perfusion flow. B. ATP response of an oocyte expressing hP2X1 receptors. Insert, magnification of the baseline showing membrane oscillations before ATP application, and their absence after ATP perfusion, calibration bars indicates values for the recording in panel B and the insert. C. Individual STICs in two oocytes recorded at 24 and 48 hrs after injection of cRNA for the hP2X1 subunit.

The STICs frequency, 24 h after injection, was 0.25 ± 0.13 Hz (mean ± SD; n = 20 oocytes) and gradually increased with time. Identification of individual events started to be difficult ≈48 hours after cRNA injection (Fig. 1C). The amplitude of individual fluctuations was highly variable and skewed to lower values (Fig. 2, mean ± SD = 15.2 ± 16.1 nA; median = 9.5 nA; range of 1 to 160 nA; n = 2086 events from 10 oocytes). The rise time to peak and the decay time constant had skewed distributions with medians of 57 ms and 125 ms respectively (n = 470 events from 10 oocytes). STICs were completely blocked by 10 μM NF023, a potent P2X1 antagonist (Soto et al., 1999), confirming their dependence on hP2X1 expression (Fig. 2C). These results indicate that STICs are generated by vesicular release of ATP and autocrine hP2X1 receptor activation.

Fig 2.

Fig 2.

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STICs are dependent on the activation of hP2X1 receptors. A. STICs in an oocyte injected with cRNA for hP2X1 in resting conditions and voltage clamped at −80 mV. B. Distribution of STICs amplitude in 10 oocytes. Median = 9.5 nA. Insert, amplification of a STIC from the oocyte shown in A (arrow). C. Blocking of STICs and reduction of the holding current (open conductance) by NF023, a P2X1 antagonist. D. Apyrase, which degrades ATP, also reduced the STICs amplitude and the holding current. E. Scanning Electron Microscope (SEM) photograph of a fixed oocyte that had STICs and was completely devoid of follicular cells on its surface. Xenopus oocytes were processed as previously described (Miledi and Woodward, 1989) and photographed using a FEI Quanta 3D FEG Dual Beam (SEM/FIB) microscope at 10KV.

Interestingly, NF023 also reduced the holding current by 37 ± 21.9 nA (n = 8 oocytes). The decrease in the tonic current was associated to a conductance reduction of 798 ± 459 nS, measured with a 20-mV pulse before and during NF023 perfusion (mean ± SD; n = 7 oocytes). The enzyme apyrase (5 U/ml), that hydrolyses ATP, decreased the holding current by 44 ± 9.8 nA (mean ± SD; n = 3 oocytes) confirming its dependence on extracellular ATP (Fig. 2D). Therefore, ATP not only triggers STICs but also is able to sustain a tonic conductance through continuous activation of hP2X1 receptors on the membrane of the oocyte.

It is known that Xenopus oocytes release ATP (Maroto and Hamill, 2001; Nakamura and Strittmatter, 1996) that activates purinergic receptors on follicular cells covering their surface (Saldana et al., 2009; Saldana et al., 2005); however, it is not clear if follicular cells release ATP themselves. To discard the possibility that follicular cells were releasing ATP, oocytes were first rolled on Petri dishes treated with poly-D-lysine, to ensure complete removal of follicular cells (Miledi and Woodward, 1989), before injection with cRNA for hP2X1. Oocytes in which the absence of follicular cells was confirmed by Scanning Electron Microscopy (Fig. 2E) also had spontaneous fluctuations and a tonic conductance, both blocked by NF023 (n = 6 oocytes), indicating that STICs originated from ATP released from the oocyte at rest.

Extracellular and intracellular Ca2+ on STICs and tonic current

To determine the role of extracellular Ca2+ we substituted Ba2+ for Ca2+ in the extracellular solution. Figure 3 shows that Ba2+ reduced the amplitude and frequency of STICs as well as the tonic current. Due to the complex nature of the signal and the difficulty to separate STICs from the tonic current we decided to determine the amount of inward current that was driven by hP2X1. For this, we calculated the mean value of the cumulative distribution, point by point, of NF023-sensitive current in 60 seconds epochs (Fig. 3). Interestingly, nearly 80% of the current sensitive to NF023 was reduced by using Ba2+ instead of Ca2+ (n = 21 oocytes). Because Ba2+ permeates P2X1 channels, but has low efficacy when activating endogenous CaCCs (Oh et al., 2008) it follows that a large proportion of the ATP induced-current (STICs and tonic) depends on the activation of endogenous CaCCs, which is in agreement with the high Ca2+ permeability of P2X1 channels (Evans et al., 1996; Samways et al., 2014). Furthermore, the inversion potential for STICs was near the equilibrium potential for Cl− in normal Ringer with Ca2+ (Fig. 4A, B; ESTICs ≈ −20 mV)(Kusano et al., 1982). However, the inversion potential was shifted to depolarized values when using Ringer with Ba2+, as inferred from the continuous appearance of inward fluctuations at −20 and −10 mV (Fig. 4C). The frequency and amplitude of STICs was voltage-dependent; STICs were enhanced by hyperpolarizing voltages but were reduced at depolarizing ones; therefore, the presence of STICs in Ba2+ at depolarized values was not reliable enough to precisely determine their inversion potential.

Fig. 3.

Fig. 3.

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Effects of Ba2+ substitution on STICs and tonic current. A. Membrane oscillations of an oocyte expressing hP2X1 receptors at rest in normal Ringer’s solution, in a solution where Ba2+ replaced Ca2+, and during the application of NF023. The oocyte was voltage clamped at −80 mV. Notice that returning to normal Ringer produced a rebound of STICs and tonic current. The current resistant to NF023 was used as the baseline (zero) for current measurements B. Cumulative distribution of the inward current sensitive to Ba2+ and NF023 in 60 seconds epochs. C. Normalized current, at the 0.5 value of the cumulative distribution, for the different conditions shown in A (n = 21 oocytes). **, p<0.0005; *** p<0.0001, Oneway ANOVA followed by multiple comparisons vs Ringer’s solution control using Dunnett’s method.

Fig. 4.

Fig. 4.

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Effects of voltage on STICs. A. Representative traces of an oocyte at rest, clamped at different voltages, before and during the perfusion of NF023. B. Plot of the change in the frequency of STICs elicited at different voltages normalized to the STICs frequency at −80 mV. Notice the absence of membrane oscillations around −20 mV (n = 8 oocytes). C. membrane oscillations at values more depolarized than the equilibrium potential for chloride, using Ringer with Ca2+ or Ba2+ in the same oocyte. Notice that inward fluctuations (arrows) can be followed by outward fluctuations (asterisks) when Ca2+is present in the extracellular solution. When Ba2+ is used instead of Ca2+ only inward fluctuations are observed.

To discard the possibility that NF023 was non-specifically blocking CaCCs, we tested the effects of NF023 on the membrane oscillations elicited by 1:1000 rabbit serum (RS). These oscillations are dependent on the opening of CaCCs in the membrane of the oocyte (Tigyi et al., 1990). We found that NF023 had no effect on the amplitude of the oscillatory responses (Fig. 5) providing evidence that the reduction of the tonic response is due to the blockade of hP2X1 receptors and not of CaCCs (n = 6 oocytes).

Fig. 5.

Fig. 5.

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Effects of NF023 on CaCCs. A. Perfusion of 10 μM NF023 to non-injected oocytes voltage-clamped to −80 mV did not modify (B) CaCCs-dependent membrane oscillations triggered by 1:1000 rabbit serum (RS) (n = 6).

Non-injected oocytes may spontaneously generate membrane fluctuations. However, the proportion of oocytes with spontaneous oscillations account for less than 20% of all oocytes in a single experiment (Berridge, 1991; Kusano et al., 1982). The fluctuations depend on Ca2+ released from intracellular stores. By injecting 0.5 M EGTA in oocytes expressing hP2X1 or incubating them within 50 μM of a membrane permeable calcium chelator (BAPTA-AM) we reduced the mean value amplitude of NF023 sensitive current by 63% (two tails, t-test, p = 0.002 n =26 oocytes). These methods did not completely prevent the appearance of STICs (Fig. 6), indicating a partial dependence on intracellular Ca2+.

Fig. 6.

Fig. 6.

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Effects of BAPTA-AM on STICs and tonic current. A. The efficacy of 50 μM BAPTA-AM to chelate intracellular Ca2+ was monitored by the complete blockade of Ca2+ oscillations induced by 1:1000 RS. B. Membrane oscillations of an unstimulated oocyte expressing hP2X1 receptors and incubated overnight in 50 μM BAPTA-AM. Notice that presence of STICs in Ringer with Ca2+ and the strong reduction in amplitude when Ba2+replaced Ca2+. The current resistant to NF023 was used as the baseline (zero) for current measurements. C. Cumulative distribution of the inward current sensitive to Ba2+ and NF023 in 60 seconds epochs. D. Normalized current, at the 0.5 value of the cumulative distribution, for the different conditions shown in A (n = 10 oocytes). *, p<0.01; ** p<0.05, Oneway ANOVA followed by multiple comparisons vs Ringer’s solution control using Dunnett’s method.

The presence of STICs in oocytes expressing hP2X1 receptors at rest were independent of extracellular Ca2+ and partially dependent on intracellular Ca2+. Therefore, it is possible that the basal vesicular release of ATP that elicits the STICs is a constitutive phenomenon of the oocyte. This is in agreement with a hypothesis posed by Maroto and Hamill (Maroto and Hamill, 2001) that ATP in these cells is released during the transport of vesicle trafficking from the Golgi delivering protein cargo to the cell membrane. To test this hypothesis, we incubated oocytes expressing hP2X1 in 20 μM brefeldin, a blocker of intracellular vesicular trafficking and ATP release from the oocyte (Maroto and Hamill, 2001). Brefeldin treatment eliminated the presence of STICs providing strong evidence for this hypothesis (n = 5). Interestingly, brefeldin did not completely block the Ba2+- and NF023-sensitive tonic current, even in BAPTA-AM treated oocytes (Fig. 7). In brefeldin treated oocytes restoring the extracellular Ca2+ elicited a fast-inward current that was reduced 10-fold by BAPTA-AM (Fig. 7B), suggesting the presence of Ca2+-permeable pathway continuously open that, when Ca2+ is reintroduced in the extracellular solution, activates CaCCs. To determine whether CaCCs amplify the effects of low concentrations of ATP on hP2X1 activation we tested the effects of 300 pM ATP, a concentration with no known effects on wild type homomeric P2X1 channels. Substitution of Ba2+ for Ca2+ in the Ringer’s solution masked the high sensitivity of hP2X1 channels to 300 pM ATP (Fig. 8). Taken together these results strongly suggests that Ca2+-activated chloride channels amplify the non-desensitizing component of hP2X1 channels activated by spontaneous release of ATP from Xenopus oocytes.

Fig. 7.

Fig. 7.

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Effects of brefeldin incubation on STICs. A. Representative current trace of oocytes expressing hP2X1 receptors after overnight incubation in 50 μM BAPTA-AM and 3 hours treatment in 20 μM brefeldin. In this condition oocytes did not show STICs, before, during, or after perfusion with Ba2+ solution. A persistent tonic current is still present as well as the current rebound after Ba2+ (arrow). B. Comparison between oocytes treated with brefeldin, with and without incubation with BAPTA-AM; ** p<0.001, Mann-Whitney U test (n = 5; each group)

Fig. 8.

Fig. 8.

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Amplification of ATP induced signal inXenopus oocytes. Tonic currents elicited by 300 pM ATP are eliminated by the replacing Ba2+ for Ca2+ in the extracellular solution. Notice that small changes in flow produce mechanical artifacts due to vesicular release of ATP (n = 3).

Discussion

ATP release from Xenopus oocytes

ATP release from oocytes at rest has been quantified by luciferin-luciferase assays (Maroto and Hamill, 2001). While the strength of this method is its sensitivity in the femtomolar range, it only measures ATP in bulk solutions and lacks the resolution to visualize individual events triggered by vesicular release. P2X1 receptors, on the other hand, have been used to detect Ca2+-dependent exocytic release in rat chromaffin cells (Hollins and Ikeda, 1997), demonstrating the possibility to use P2X1 receptors as an ATP sensor. The use of P2X1 has allowed the study of single release events (Fabbro et al., 2004) to the large vesicular release observed during mechanical stimulation (Saldana et al., 2009). Here, by using P2X1 receptors we demonstrate that Xenopus oocytes release packages of ATP via a brefeldin-dependent vesicular mechanism. Such vesicular release could be monitored by the presence of STICs which were mediated by the autocrine activation of the heterologous expressed hP2X1 receptors and amplified by the activation of endogenous CaCCs. Spontaneous STICs were independent of extracellular Ca2+ and partially independent of intracellular Ca2+ suggesting that the basal vesicular release of ATP is a constitutive phenomenon of the oocyte. This is in agreement with the previous hypothesis posed by Maroto and Hamill (Maroto and Hamill, 2001) stating that ATP in Xenopus oocytes is released during the transport of vesicle trafficking from the Golgi delivering protein cargo to the cell membrane. Vesicular release from the oocyte seems to contribute, at least partially, to the persistent activation of hP2X1 and CaCCs. After removal of intracellular and extracellular sources of Ca2+ and blockade of vesicular transport, there was a small component of persistent activity of hP2X1. Possible explanations for this persistent activity could be that the complex topology of the oocyte’s membrane (Zampighi et al., 1995) slows the diffusion of ATP outside the microvilli after vesicular release, ATP is continuously flowing through hemichannels formed by connexins Cx38 (Bahima et al., 2006), or that at any given time a small percentage of hP2X1 receptors is open in absence of ATP. These explanations are not mutually exclusive and could be acting synergistically.

It has been shown that a stable number of heterologously expressed proteins in the oocyte’s membrane is reached at around 72 h post injection of cRNA (Zampighi et al., 1999). Therefore, the continuous increase of STICs during the first 3 days after injection is more consistent with the continuous insertion of new hP2X1 receptors and not with an increase in the rate of vesicular fusion. Similarly, the increase in the NF023-sensitive tonic current would result from a larger number of hP2X1 being activated by low concentrations of ATP, either by slow diffusion or ATP leakage through hemichannels (Bahima et al., 2006). Rettinger and Schmalzing (Rettinger and Schmalzing, 2003) have shown that ATP concentrations as low as 3 nM induce 50% of steady state desensitization of rat P2X1 receptors. For this reason, it is likely that the tonic NF023-sensitive current observed in presence of extracellular Ca2+ is carried by a small percentage of non-desensitized hP2X1 receptors and amplified by endogenous CaCCs, and carried only by non-desensitized hP2X1 receptors when all sources of Ca2+ have been removed (e.g. using 50 μM BAPTA-AM incubation and extracellular Ba2+ solutions). Temporal overlap of activated hP2X1 at different ATP release sites and/or Ca2+-release Ca2+ mechanisms (Shuai et al., 2006) could also contribute to the tonic activation of CaCCs. Interestingly, deacceleration of ion current decay at low concentrations has been observed in P2X3 receptors (Grote et al., 2008). Because P2X1 and P2X3 receptors share many kinetic characteristics, a slower decay of hP2X1 may be a factor in the generation of the NF023-sensitive tonic current.

Previous studies have observed that ATP release, measured by luciferase assay, is increased by hyperpolarizing pulses, but the exocytotic release of ATP was ruled out because the release was independent of extracellular Ca2+ (Bodas et al., 2000). Our experiments indicate that hyperpolarizing voltages increase the STICs frequency which could lead to an increase in ATP previously observed but because it is a constitutive phenomenon is independent of extracellular Ca2+. The release of ATP from the oocyte should have important physiological consequences in the paracrine activation of purinergic receptors in follicular cells covering the oocyte, including the activation of ATP-sensitive K+ channels, the modulation of cAMP-dependent K+ currents and G protein coupled purinergic receptors (Arellano et al., 2009; Saldana et al., 2009; Saldana et al., 2005).

Functional relevance of CaCCs and P2X1-mediated tonic activity

Our results indicate that ATP release from the oocyte is able to sustain a continuous activity of hP2X1 receptors, and this activity is in turn greatly amplified by its coupling with CaCCs. Similarly, vascular smooth muscle cells express CaCCs and P2X1 receptors, which are important in vascular contractility and regulation of blood pressure (Heinze et al., 2014), and release ATP during mechanical stimulation produced by shear stress (Lohman et al., 2012); however, how a highly desensitizing receptor is able to maintain the vascular tone is not known. Interestingly, P2X1 receptors and CaCCs have a non-random pattern of expression in vascular smooth cells. CaCCs are localized to lipid rafts as shown by the receptors colocalization with lipid raft marker caveolin-1 (Jin et al., 2013). Similarly, P2X1 receptors are colocalized with caveolin-1 in rat vas deferens and bladder smooth muscle cells (Vial et al., 2006). The co-localization of P2X1 and CaCCs receptors to lipid rafts potentially allows for a more localized physiologic reaction. It has also been shown that CaCCs control smooth muscle contractility in cerebral arteries in response to pressure induced activation (Bulley et al., 2012) hinting at a possible connection between shear stressed induced ATP release, P2X1 activation, and CaCCs-induced smooth muscle contraction. Future studies directly addressing the relationship between hP2X1 receptors and CaCCs should provide information about the mechanisms that regulate the vascular tone, and a potential target for pharmacological modulation in cardiovascular disease.

Highlights:

  • Ca2+-independent vesicular release of ATP in Xenopus oocytes

  • Vesicular release of ATP is blocked by brefeldin.

  • ATP release can persistently activate hP2X1 receptors

  • ATP-dependent tonic activity of hP2X1 is amplified by Ca2+-activated Cl− channels

Acknowledgments

Funding for this project was partially provided by the University of California Institute for Mexico and the United States (UC-MEXUS; CN-13-613) to AL and RM; the King Abdul Aziz City for Science and Technology, Saudi Arabia [Grant KACST-46749] to RM; and the National Institute of Health (R21MH113177) to AL. The authors thank M.Sc. Dhatri Pandya for her technical help in some experiments.

Footnotes

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