P2Z purinoceptor-associated pores induced by extracellular ATP in macrophages and J774 cells

Robson Coutinho-Silva and Pedro Muanis Persechini

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-900 Rio de Janeiro, Brasil

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Millimolar concentrations of extracellular ATP (ATPo) can induce the permeabilization of plasma membranes of macrophages and other bone marrow-derived cells to low-molecular-weight solutes, a phenomenon that is the hallmark of P2Z purinoceptors. However, patch-clamp and whole cell electrophysiological experiments have so far failed to demonstrate the existence of any ATPo-induced P2Z-associated pores underlying this permeabilization phenomenon. Here, we describe ATPo-induced pores of 409 ± 33 pS recorded using cell-attached patch-clamp experiments performed in macrophages and J774 cells. These pores are voltage dependent and display several properties of the P2Z-associated permeabilization phenomenon: they are permeable to both large cations and anions, such as tris(hydroxymethyl)aminomethane, N-methyl-D-glucamine, and glutamate; their opening is favored at temperatures higher than 30°C; they are blocked by oxidized ATP and Mg2+; and they can be triggered by 3'-O-(4-benzoylbenzoyl)-ATP but not by UTP or ADP. We conclude that the pores described in this report are associated with the P2Z permeabilization phenomenon.

adenosine 5'-triphosphate; permeabilization; 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate; oxidized adenosine 5'-triphosphate; uridine 5'-triphosphate; P2X7

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

IT HAS BEEN WELL ESTABLISHED that extracellular ATP (ATPo) may trigger intracellular signaling pathways, open ion channels, and induce different physiological responses depending on cell type and P2 purinoceptors expressed [reviewed by Dubyak and El-Moatassim (12)]. Five types of receptors, termed P2Y, P2U, P2X, P2T, and P2Z, have been identified based on pharmacological and functional studies (7, 15). Recently, based mainly on the analysis of protein sequences and signal transduction mechanisms, a new nomenclature scheme has been proposed for these receptors (5, 15, 36). According to this view, there are two major families of P2 purinoceptors, one with the properties of intrinsic ion channels, termed P2X, and the other coupled to G proteins, termed P2Y.

The term P2Z continues to be used to name receptors associated with the opening by ATP4- of a nonselective, poorly characterized ion pore (11, 15). Its presence has been described in many tissues and systems, including bone marrow-derived cells such as macrophages, mast cells, thymocytes, some lymphocytes, the phagocytic cells of the thymic reticulum (PT-R cells), and Langerhans cells (9, 11, 27, 38, 39). P2Z receptors have been frequently detected indirectly by the permeabilization of the cell membrane to fluorescent dyes such as lucifer yellow and ethidium bromide that happens a few minutes after ATPo addition (34, 39). Macrophages and mast cells are permeable to solutes of up to 900 Da, whereas some lymphocytes, thymocytes, and hematopoietic stem cells are permeable to solutes of up to 400 Da (12, 15, 27).

The physiological function of P2Z purinoceptors in the immune system is still an open question (11, 12, 29). In macrophages, it has been associated with interleukin-1 maturation and release (21), formation of multinucleated giant cells (14), and elimination of macrophages infected by intracellular parasites (22). However, due to the strong permeabilization phenomenon and the induction of apoptosis in some cell types such as thymocytes and macrophages, a role in cell death has been proposed (11).

Patch-clamp studies have generated valuable information regarding the interaction of ATP with P2Z purinoceptors. However, experiments performed in mast cells (38) and macrophages (1, 6, 18) have so far failed to detect single-channel currents that could be associated with an ATPo-induced pore. In macrophages and PT-R cells, two currents can be promptly induced by ATPo: a depolarizing current that is selective for small monovalent cations and a Ca2+-dependent K+ current (1, 9, 17). We have recently shown that this depolarizing current can be ascribed to a 5- to 8-pS channel that is too small to explain the permeabilization phenomenon (10). Moreover, the proposal of involvement of hemi-gap junction channels formed by connexin-43 (3) could not be confirmed (2).

To further investigate the nature of the P2Z-associated permeabilization phenomenon, we performed experiments using the cell-attached configuration of the patch-clamp technique in macrophages under conditions known to induce permeabilization. These experimental conditions would avoid modifications of the intracellular environment and increase our chances of obtaining direct electrophysiological recordings of a putative P2Z pore. Here, we describe for the first time single-channel currents of large nonselective channels opened by ATPo in mouse peritoneal macrophages and J774 cells. The conductance, selectivity, and pharmacological characteristics of these pores are consistent with the expected properties of a P2Z-associated pore.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells. Thioglycolate-elicited macrophages were obtained from the intraperitoneal cavity of Swiss-Webster mice. Cells were transferred to RPMI 1640 medium containing 5% heat-inactivated fetal calf serum, 2 g/l sodium bicarbonate, 100 U/ml penicillin, and 100 µg/ml streptomycin and were plated in 35-mm petri dishes. All surgical manipulations were performed under ether anesthesia. After 1 h of incubation at 37°C in a 5% CO2 humidified atmosphere, nonadherent cells were removed, and the adherent cells were kept in the same conditions for 4 h to 15 days until use. Unless otherwise specified, we used macrophages in our experiments. In some experiments, the mouse macrophage J774 cell line (J774 cells) was used. The cells were grown in 25-ml tissue culture flasks kept at the same conditions as above and were plated in 35-mm petri dishes for 2 h to 4 days before use.

Reagents. ATP, UTP, ADP, 3'-O-(4-benzoylbenzoyl)-ATP (BzATP), oxidized ATP, ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), tris(hydroxymethyl)aminomethane (Tris), N-methyl-D-glucamine (NMDG), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and sodium glutamate were purchased from Sigma Chemical (St. Louis, MO). The pore-forming protein of cytotoxic lymphocytes (perforin) was purified from the CTLL-R8 cell line by low-pressure liquid chromatography using a Q-Sepharose column coupled to an fast-performance liquid chromatography system (Pharmacia Biotech, Uppsala, Sweden) as previously described (20, 30). Perforin activity (5 hemolytic U/µl) was quantified in a Ca2+-dependent hemolytic assay as described previously (20).

Electrophysiological measurements. Ionic currents were studied in cell-attached and, in some cases, whole cell configuration, using an EPC-7 amplifier (List Electronic, Darmstadt, Germany) according to standard patch-clamping techniques (16). The petri dishes containing the cells were filled with 10 ml of solution and were placed in the heated stage of a microscope. Gigaohm seals were formed after offset potential compensation, using heat-polished micropipettes of 5-10 MOmega . ATP, UTP, and ADP (100 mM) were applied to 10 ml of extracellular solution by manually dropping 50-100 µl of solution into the plate dish. BzATP (100 µl, 14 mM) was applied to 7 ml of extracellular solution. All drugs were diluted in normal extracellular solution and were stored in the dark at -20°C until use. Temperature was continuously monitored with a digital thermometer placed in the extracellular solution and, unless otherwise specified, was maintained in the range of 30-37°C.

Current and voltage were simultaneously recorded on a paper chart recorder (Mark VII WR 3310; Graphtec, Yokohama, Japan) and in a VCR tape, and digitalization was performed by a Neurocorder (model DR-390; Neuro Data Instruments) for off-line analysis. Conductances of unitary channels were obtained by manually fitting a straight line throughout the current value corresponding to the open state of neighbor events and then measuring the amplitude. Current-voltage (I-V) curves were then plotted, and linear regressions were performed using the SigmaPlot software (Jandel). Figures 1-6 are representative records of each type of experiment described in the text. I-V plots in Figs. 1-3 were taken from single channels of the same patches. Mean values and the number of experiments (n) are described in the text and in Table 1. Current signals were filtered at 3 kHz during acquisition and at 300 Hz during the play back of the recordings. To correct for junction potentials, the ground electrode was placed in a chamber containing the same solution as the patch pipette and was connected to the extracellular solution by an agar bridge. The diffusion potentials at the tip of the patch pipette were then measured for each intrapipette solution at the beginning of each experiment, as described by Neher (26). All voltages shown in Figs. 1-6 refer to the holding potentials (VH) inside the patch pipette, without any corrections, whereas the numeric values of the reversal potentials (VRev) shown in the text and in Table 1 are corrected for the pipette junction potential. Average data are given as means ± SD.

Unless otherwise specified, the compositions of the extracellular and intrapipette solutions were the same (in mM): 150 NaCl, 5 KCl, 1 MgCl2, and 10 Na-HEPES, pH 7.4 (normal extracellular solution). In the whole cell experiments, the intrapipette solution contained (in mM) 150 KCl, 5 NaCl, 1 MgCl2, 0.1 K2-EGTA, and 10 K-HEPES, pH 7.2 (normal intracellular solution). Ion substitution experiments were performed by using the following intrapipette solutions: in low-Na+ solutions, 150 mM of either Tris · Cl or NMDG-Cl substituted for NaCl; in low-Cl- solution, 150 mM sodium glutamate substituted for NaCl. Low NaCl contained (in mM) 34 NaCl, 158 mannitol, 0.12 CaCl2, 0.5 MgCl2, 1.4 EGTA, and 5 HEPES, pH 7.4. Typical junction potentials were 4, -5.5, and -3.5 mV for low-Na+, low-Cl-, and low-NaCl solutions, respectively. Normal extracellular solution did not require any correction.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Single-channel currents induced by ATPo in cell-attached patches. Because previous patch-clamp experiments have failed to detect P2Z-associated pores (1, 18, 24, 25), we decided to investigate the occurrence of any ATPo-triggered ion channel in cell-attached patches at 30-37°C using normal extracellular solution both in the patch pipette and in the extracellular bath. These conditions avoided any disturbance to the intracellular medium and assured that most macrophages would become permeable to lucifer yellow upon ATP addition. Moreover, because the transduction signals involved in the permeabilization phenomenon may require second messengers (4), ATP was added to the extracellular solution only after gigaseal formation. As shown in Fig. 1A, addition of normal extracellular solution in the proximity of the cell did not induce any ion channel activity on the patch, whereas addition of ATP induced the opening of several ion channels. At pipette VH ranging from -20 to -60 mV, larger steps of current were frequently observed. To evaluate channel conductances and VRev, the resting transmembrane potential of the cell was measured in independent current-clamp whole cell experiments performed at least 30 s after addition of ATPo. Using normal intracellular solution in the pipette, we obtained a value of -1 ± 2 mV (n = 6), consistent with the already described depolarization induced by ATPo (1, 6, 18).


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Fig. 1.   Single-channel activity induced by extracellular ATP (ATPo) in cell-attached patches of macrophages and J774 cells. Experiments were performed at 30-37°C, using normal extracellular solution inside the pipette. A: typical recording from a macrophage indicating that addition of normal extracellular solution (first two vertical arrows) did not cause any effect, whereas addition of ATP (third vertical arrow) caused an inward current and unitary channel activity followed by the opening of large current steps. Holding pipette potential (VH) was -30 mV. Large-conductance channels are recorded at different VH values in a macrophage (B) and in a J774 cell (C). Correspondent current-voltage (I-V) plots are shown in D. Conductance and reversal potentials (VRev), calculated by linear regression, were 402 pS and 0.2 mV for the macrophage and 422 pS and -0.7 mV for the J774 cell. Final concentration of ATPo was 500 µM in all experiments. Horizontal arrows on left indicate I = 0 pA, and values of potential on right of each record refer to VH, without correction of junction potentials.

A preliminary evaluation of our data showed that the observed channels belonged to at least two categories: those displaying VRev compatible with K+ channels and those with VRev close to 0 mV and conductances larger than 300 pS. We have therefore decided to focus our attention on the latter. To decrease the presence of unrelated channels, we have always waited 3-5 min after obtaining the cell-attached seal before applying ATP. Patches displaying spontaneous channel activity were discarded. In some experiments, we also added 2.5 mM Ba2+ to both intrapipette and extracellular solutions to block K+ channels. However, the use of ion channel inhibitors was avoided because we were looking for a pore of unknown properties and we did not want to disturb any of the cell characteristics at this stage of the investigation.

Figure 1, B and C, illustrates the opening of large channels observed at different VH in a macrophage and in a J774 cell, respectively. I-V plots of these channels are shown in Fig. 1D. The mean conductance and VRev values measured in seven different macrophages were 409 ± 33 pS and -2 ± 2 mV, respectively (Table 1). Similar values were obtained for J774 cells (409 ± 64 pS, 2 ± 3 mV, n = 3). Addition of 2.5 mM BaCl2 to both intrapipette and extracellular solutions did not significantly change these values (468 ± 52 pS, -0.6 ± 0.6 mV, n = 3).

                              
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Table 1.   Reversal potential and conductance of ATPo-induced pore

We have observed channel activity similar to that described in Fig. 1 in 87% of cell-attached patches (n = 80). The first signs of channel activity were detected 10-180 s after ATPo application. Frequently, two or three channels stayed opened at the same time. Opening times were widely scattered, varying from 60 ms to 30 s, and were dependent on membrane potential, as shown later (e.g., see Fig. 5).

The fact that ATP permeabilizes the whole macrophage membrane, inducing osmotic stress and disruption of the intracellular homeostasis, raises the question of whether the pores we have described are triggered by specific P2Z-associated signaling cascades or represent a nonspecific effect that could be activated even by other nonnucleotide permeabilizing agents. To address this question, we added perforin to macrophages during cell-attached experiments performed under the same conditions used for ATP. This pore-forming protein is derived from cytotoxic lymphocytes and can open large pores ranging from 400 pS to 6 nS in the membranes of many cell types (31). Pore insertion takes <1 min; therefore, the time course of permeabilization by perforin is in the same order of magnitude as by ATP. Addition of 250 hemolytic units of perforin to cell-attached macrophages (n = 6) did not induce the opening of any large-conductance channels in the macrophage patches (data not shown). However, a pattern of cell blebbing, typical of perforin pore formation, could easily be observed in the neighboring cells within 1-2 min, as previously described (31). Pore formation by perforin was further confirmed in whole cell recordings in which a series of step-like conductance increases typical of perforin pore formation was observed a few seconds after perforin addition (data not shown). Moreover, in another series of experiments, perforin was shown to induce permeabilization of macrophages to ethidium bromide and lucifer yellow (data not shown). These results demonstrate that perforin induces pore formation only in the portion of membrane situated outside the cell-attached patch. Our data are consistent with the interpretation that the ATP-induced pores are triggered by an ATP-specific mechanism and not by other mechanisms induced by unrelated permeabilizing agents.

Selectivity of ATPo-induced channels. The size and the VRev of the channels described in Fig. 1 suggested that they could be nonselective, allowing the passage of both cations and anions of different sizes, as it would be expected for a pore involved in the P2Z-associated permeabilization phenomenon. To investigate this question, we have performed cell-attached ion substitution experiments using four intrapipette solutions containing different concentrations of cations and anions. Junction potentials were taken into account as described in MATERIALS AND METHODS. In all solutions, we observed large channels displaying VRev close to 0 mV, but, in some conditions, the channels had at least two different sizes (Figs. 2 and 3 and Table 1). When NMDG [relative molecular weight (Mr) of 195] substituted for Na+ (Fig. 2A), a channel of 350 ± 30 pS (n = 8) and VRev of -2.3 ± 2.5 mV were observed. When Tris (Mr 121) substituted for Na+ (Fig. 2, B, C, and E), channels with two different conductances (340 ± 20 pS, n = 3 and 221 ± 31 pS, n = 6) were observed. Both had VRev close to 0 mV and could be clearly distinguished in I-V plots (Fig. 2E and Table 1).


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Fig. 2.   Selectivity of ATPo-activated channels. Macrophage patches were studied using the following solutions inside the pipette in three independent experiments: NMDG (A), Tris (B and C), and glutamate (D). I-V plots of channels shown in B and C are shown in E. Values of conductance and VRev were 343 pS and 1.7 mV for Z-1 pores and 233 pS and -0.9 mV for Z-2 pores, respectively. Final concentration of ATPo was 500 µM in all experiments. Horizontal arrows on left indicate I = 0 pA, and values of potential on right of each record refer to VH, without correction of junction potentials.


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Fig. 3.   ATPo-activated channels are permeable to both cations and anions. Macrophage patches were studied using a low NaCl solution inside the pipette. Typical single-channel records are shown in A and B. I-V plots for a Z-1 and a Z-2 channel are shown in C. Values of conductance and VRev were 380 pS and -2.6 mV for Z-1 pores and 285 pS and -0.2 mV for Z-2 pores (after correcting for junction potentials), respectively. Horizontal arrows on left indicate I = 0 pA, and values of potential on right of each record refer to VH, without correction of junction potentials. Experimental conditions and data analysis were the same as in Fig. 1.

When glutamate (Mr 146) substituted for Cl- (Fig. 2D), the recordings were frequently unstable, but all cells tested (n = 12) responded to ATPo. We could plot I-V curves for the channel activity of three of these cells, obtaining the values of 358 ± 78 pS and 1.3 ± 1.2 mV for the conductance and the VRev, respectively.

The data described so far are consistent with channels that are nonselective for both cations and anions. However, channels selective for either cations or anions could lead to similar results. Therefore, we have decreased NaCl concentration to 34 mM in the intrapipette solution, using mannitol to maintain osmolarity, a condition that makes cation and anion channels have VRev values of opposite signs (33). As shown in Fig. 3 and in Table 1, activities of two channels with conductances of 345 ± 31 (n = 3) and 279 ± 8 (n = 4) were found. VRev values were 2 ± 7 mV (n = 3) and 5 ± 2 (n = 4) mV, respectively, leading us to the conclusion that both channels are cation and anion nonselective pores triggered by ATPo.

The finding that the above-described pores are permeable to large cations and anions such as Tris, NMDG, and glutamate suggests that they could be involved with the phenomenon of P2Z-associated permeabilization, with a size exclusion limit of at least 195 Da. Therefore, we named the larger and smaller pores Z-1 and Z-2, respectively.

Other properties consistent with a P2Z-associated pore. To further characterize the above-described pore(s) as involved in the P2Z-associated permeabilization, we investigated other known properties of this phenomenon (7, 11, 12, 23, 34): temperature dependence, triggering by BzATP, lack of effect of ADP and UTP, and blockade by Mg2+ and oxidized ATP (Fig. 4). In this series of experiments, we kept the intrapipette VH in the range from -20 to -30 mV since, under this condition at 37°C, ATPo induces pore opening in <30 s, as shown in Fig. 1.


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Fig. 4.   Properties consistent with P2Z-associated pore. A: ATP (500 µM final concentration) was applied to extracellular medium at 18°C (vertical arrow in the top trace), and patches were heated to 37°C. VH was kept at -30 mV. Membrane patch remained silent for >5 min. Temperature was then gradually raised. Later (6 min), when the temperature reached 32°C, large-conductance channel activity was observed (bottom trace); 200 µM 3'-O-(4-benzoylbenzoyl)-ATP (BzATP; B), 1 mM ADP (C), or 1 mM UTP (final concentration; D) was applied using the same conditions as ATP; E: 1 mM ATP was added to the normal extracellular medium containing 10 mM Mg2+; F: 10 mM Mg2+ (final concentration) was added to the normal extracellular medium containing 1 mM ATP; G: preincubation with 300 µM oxidized ATP for 2 h was followed by addition of 1 mM ATP as indicated; H: 1 mM ATP (final concentration) was added to the normal extracellular medium. Bottom trace represents magnified view of indicated part of the record. Vertical arrows and horizontal bar indicate drug application, and horizontal arrows indicate I = 0 pA. Values of VH, without correction of junction potentials, were -20 mV in B and -30 mV in A and C-H. Experimental conditions and data analysis were the same as in Fig. 1.

At 18-22°C, the cell-attached patches remained silent for >10 min after ATPo addition (Fig. 4A, top trace). However, upon gradual heating, Z-1 type channels could be easily observed in all cells studied (n = 5) when the temperature reached 30-37°C (Fig. 4A, bottom trace).

BzATP, a P2Z agonist that is more potent than ATP in inducing permeabilization and Ca2+ mobilization (13), was also able to open large-conductance channels with I-V curves similar to the Z-1 pores triggered by ATPo (Fig. 4B). The mean values of the conductance and VRev were 425 ± 22 pS (n = 4) and -0.8 ± 2 mV (n = 4), respectively. In addition, neither ADP (n = 6) nor UTP (n = 6) induced pore activity in the macrophage patches (Fig. 4, C and D, respectively).

Another characteristic of P2Z-associated permeabilization, the requirement of ATP4-, was investigated by the addition of Mg2+ to the extracellular solution. No pores were detected when ATP was added to cells kept in normal extracellular solution containing 10 mM Mg2+ (Fig. 4E, n = 5). Under this experimental condition, the permeabilization of macrophages to ethidium bromide was also blocked (data not shown). Furthermore, addition of MgCl2 (30-100 µl of a 1 M solution) during cell-attached recordings induced the closing of the pores previously opened by ATP within the following 40 s (Fig. 4F, n = 6).

Pore formation was also inhibited by preincubation of macrophages with 300 µM oxidized ATP for 2 h at 37°C (Fig. 4G, n = 6). In parallel experiments, we have confirmed that, under this same condition, the ATP-induced permeabilization to ethidium bromide was also inhibited (data not shown). In two of the experiments with oxidized ATP (e.g., Fig. 4G), we could detect a transient current just after ATP addition. However, this current did not correspond to the opening of large pores as the ones observed after the addition of ATPo in the absence of oxidized ATP in a similar experiment (Fig. 4H).

Regulation by voltage. The activity of Z-1 and Z-2 channels was modulated by voltage. Opening times of several seconds could be measured at negative VH values, whereas, at positive values, both pores closed. This is evident in Fig. 5 in which BzATP was applied to a macrophage. A membrane patch that would look otherwise like a leaky seal at VH -9 mV displayed successive step-like current decreases, returning to 0 pA at +30 mV (Fig. 5B ). Then, the patch remained silent for 3 min until VH was shifted back to negative values, when a Z-1 pore opened again (Fig. 5D ). Interestingly, when either mannitol or sodium glutamate, but neither Tris · Cl nor NMDG-Cl, substituted for NaCl in the pipette solution, channels did not close at negative VH and, therefore, they could only be resolved at the single channel level at positive potentials (Figs. 2D and 3). These properties were shared by Z-1 and Z-2 pores, suggesting that they have similar regulatory mechanisms.


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Fig. 5.   Regulation by voltage. A: macrophage patch displayed large-conductance single channels after addition of 200 µM BzATP (final concentration). B: same patch continued to increase conductance and was kept at VH = -9 mV for a few seconds until a ramp of potential was applied, shifting VH to +30 mV (vertical arrows). Soon after VH reached +30 mV, conductance returned to 0 in 5 successive steps. C: no channel activity was recorded in the same patch, whereas VH remained at +30 mV. D: large-conductance, Z-1 channel opened again when VH was shifted to -28 mV. Horizontal arrows on left indicate I = 0 pA, and values of potential on right of each record refer to VH, without correction of junction potentials.

Cascade pattern of channel opening. Unitary channel activity, like the ones described in Figs. 1-5, was obtained by adding concentrated ATP to the extracellular bath in doses designed to reach a final concentration smaller than 500 µM. However, when the final ATPo concentration was 1 mM or higher, it was frequent to observe rapid and successive increases in the current, eventually leading to saturation of the amplifier, a condition usually associated with seal disruption. However, in eight patches, most of which had previously displayed unitary Z-1 pore activity, we could distinguish four to seven independent steps before saturating the amplifier (Fig. 6). The whole process lasts <1 s. Due to the time course and the uncontrolled nature of these events, an accurate measurement of the VRev of these steps was not possible to obtain. However, examining the events of different cells, it is reasonable to assume that the VRev would be close to 0 mV as in the case of the Z-1 unitary channel. Under this assumption, the current steps observed using normal extracellular solution would correspond to conductances ranging from 461 to 772 pS (589 ± 128 pS, n = 8).


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Fig. 6.   Cascade of step-like conductance increase. Macrophage patch that had previously displayed large-conductance (Z-1) single-channel activity after addition of ATPo suddenly increased conductance in 7 successive steps until saturation of the recording system. Event happened 6.5 min after addition of ATPo (1 mM final concentration). Horizontal arrow on left indicates I = 0 pA, and value of potential on right refers to VH, without correction of junction potentials.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The P2Z purinoceptor-associated permeabilization phenomenon was described in macrophages and mast cells more than 10 years ago (8, 35, 38). However, all whole cell and single-channel currents described so far in these cells do not have the characteristics expected for a cation and anion nonselective pore. A protein called P2X7 has been recently cloned and displayed the pharmacological and functional characteristics of P2Z receptors when expressed in several cell lines (37). ATPo-induced membrane permeabilization and a cation current similar to the ones present in macrophages were observed in these cells, but no single channels of pores were recorded from P2X7-transfected cells (37).

In the search of possible reasons for the lack of electrophysiological data corresponding to an ATPo-induced pore, we performed cell-attached patch-clamp experiments in macrophages submitted to conditions known to induce permeabilization. Here, we describe the presence of large nonselective channels in macrophages and J774 cells. The finding that these channels are permeable to large cations and anions such as Tris, NMDG, and glutamate (Mr 121-195 Da) suggests that they are involved with the phenomenon of P2Z-associated permeabilization. Therefore, we named them Z-1 and Z-2 pores, for the larger and the smaller pores, respectively. In accordance with this conclusion is the fact that these ATPo-induced pores display several other properties of the P2Z-associated permeabilization (7, 11, 12, 34; see Fig. 4); they are temperature dependent and are not triggered by ATPo in the presence of high extracellular Mg2+ concentration, indicating the requirement for ATP4-. Moreover, BzATP, but neither ADP nor UTP, can substitute for ATP in the induction of pore activity. In addition, oxidized ATP, an inhibitor of the permeabilization phenomenon, also inhibits pore activity. Taken together, our results lead to the conclusion that Z pores are involved in the P2Z-associated permeabilization phenomenon. However, more experiments are needed to establish the size limit of the molecules that can diffuse through these pores and compare it with the know values of the permeabilization phenomenon.

In several membrane patches, other channels could be observed under conditions in which Z pores were not opened (e.g., Fig. 4G). This observation could be explained by the activation of ATP-induced channels via other P2 receptors present in the macrophage membrane. Two possibilities are the activation of Ca2+-dependent K+ channels (1, 17) and the ATP-activated Ca2+-permeable channel described by Naumov et al. (24, 25). In this regard, it should be noticed that oxidized ATP does not inhibit the ATP-induced increase of the intracellular Ca2+ concentration (23).

Only Z-1 pores were clearly present when normal extracellular solution was used in the patch pipette (Fig. 1 and Table 1). Its conductance (409 pS) is in accordance with the expected value of a pore permeable to lucifer yellow (Mr of 457 and Stokes radius of 7.8 A; see Refs. 32, 38). Z-2 pores were more evident in solutions with low Na+ and/or low Cl- concentration. However, although data were not always enough to plot I-V curves, single-channel activity or steps of current compatible with Z-2 pores were also observed under all conditions studied here (data not shown). It is not clear at the moment whether Z-2 represents an independent channel or a subconductance state of the Z-1 pore. However, the existence of two P2Z-associated pores or opening states is consistent with data showing that, although macrophages are permeable to molecules of up to 900, some lymphocytes seem to have a molecular weight cut-off of ~400.

The observation that the Z pores opened in membrane patches isolated from the ATP-containing extracellular medium by the gigaohm seals indicates that these pores are coupled to purinoceptors by a pathway involving second messengers. This conclusion is consistent with previous experiments that failed to obtain large conductance steps in whole cell experiments in which the intracellular milieu was not preserved (1, 18). We have not elucidated the nature of the second messengers involved in the opening of Z pores. However, it is interesting to note that recent evidence suggests the involvement of calmodulin and phospholipase D in the permeabilization phenomenon (4, 19, 28).

On the other hand, it has been demonstrated that the P2Z/P2X7 receptor is a ligand-gated channel associated with a cation current that displays a single channel conductance of 5-8 pS, not directly involved in the transport of low-molecular-weight solutes (1, 10, 12, 37). These results can be conciliated with the second-messenger hypothesis by proposing that, although Z pores are activated by the P2Z/P2X7 receptor, they are distinct channel proteins. Alternatively, Z pores could be a new (second messenger-dependent) activation state of the P2Z/P2X7 receptor itself.

There is indeed some evidence in the literature indicating that permeabilization can be separated from other P2Z-associated phenomenon: differential activation of the cation current and the nonselective pores can be achieved in Xenopus oocytes expressing macrophage mRNA (28); calmodulin antagonists are able to prevent the lytic effects of ATPo without affecting calcium influx and membrane depolarization (4); and, in P2X7-transfected cells, permeabilization, but not the cation current, is dependent on the cytoplasmatic tail of the P2X7 protein (37). The full elucidation of this problem will require the identification of the intracellular pathways triggered by P2Z receptors and the cloning of the permeabilization pore(s).

One interesting property of Z-1 and Z-2 pores is that they tend to be closed at negative transmembrane potential (positive VH). This finding suggests that, in macrophages, they can be regulated by the balance of two opposing mechanisms also induced by ATPo: the fast depolarization caused by the small cation channels recently described by us (10) and the delayed and transitory Ca2+-dependent K+ current that follows the first one (1, 17, 29). These same mechanisms would also regulate the P2Z-associated permeabilization phenomenon. In this regard, it is interesting to notice that high extracellular K+ concentration, a condition that depolarizes the cells, enhances permeabilization in peripheral blood lymphocytes (Ref. 39 and our unpublished observations).

The cascade of steps shown in Fig. 6 is possibly associated with Z-1 pores and permeabilization. However, more data are needed to clearly establish this connection. The explosive nature of the events suggests a cooperative phenomenon in which the opening of a first pore facilitates the opening of the next ones. This pattern of conductance increase has already been described for the insertion of pores of perforin in cell membranes (31).

Our results suggest that the Z pores described in this report are triggered by P2Z purinoceptors and are involved in the permeabilization of the macrophage plasma membrane to low-molecular-weight solutes. The study of these pores may contribute to the understanding of the mechanism and functional role of plasma membrane permeabilization induced by ATPo in macrophages and other cells.

    ACKNOWLEDGEMENTS

We are grateful to Vandir da Costa for continuous technical support, to Andreia Lamoglia de Souza for helping to purify perforin, and to Drs. Luiz A. Alves and Masako O. Masuda for critical reviews of the manuscript and helpful discussions.

    FOOTNOTES

This work was financed by grants from Conselho Nacional de Desenvolvimento Cientifico e Technológico do Brasil, Financiadora de Estudos e Projetos, and Fundaçao de Amparo à Pesquisa do Estado do Rio de Janeiro.

Address for reprint requests: P. M. Persechini, Laboratório de Imunobiofísica, Instituto de Biofísica Carlos Chagas Filho da UFRJ, Bloco G do CCS; Ilha do Fundão, 21941-900 Rio de Janeiro, Brazil.

Received 9 January 1997; accepted in final form 8 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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