Modulatory activity of extracellular H⁺ and Zn²⁺ on ATP-responses at rP2X₁ and rP2X₃ receptors

Abstract

1. The modulatory activity of extracellular H⁺ and Zn²⁺ was examined on ATP-responses at rat P2X₁ (rP2X₁) and rat P2X₃ (rP2X₃) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions.

2. Superfused ATP (0.03–30 μM, at pH 7.5) evoked inward currents at rP2X₁ receptors (EC₅₀ value, 300±7 nM). ATP potency was reduced 2 fold at pH 6.5, and 6 fold at pH 5.5, without altering the maximum ATP effect. Alkaline conditions (pH 8.0) did not alter ATP activity.

3. Superfused ATP (0.01–300 μM, at pH 7.5) evoked inward currents at rP2X₃ receptors (EC₅₀ value, 1.8±0.3 μM). ATP activity was affected only at pH 5.5, reducing agonist potency 15 fold without altering the maximum ATP effect.

4. Extracellular Zn²⁺ inhibited ATP-responses at rP2X₁ receptors in a time-dependent manner, a 20 min pre-incubation being optimal (IC₅₀ value, 1.0±0.2 μM). However, the Zn²⁺ effect was pH-independent, suggesting Zn²⁺- and H⁺-inhibition of ATP-responses occur through independent processes.

5. Extracellular Zn²⁺ weakly potentiated ATP-responses at rP2X₃ receptors (EC₅₀ value, 11±1 μM). The Zn²⁺ effect was dependent on pre-incubation time and, with 20 min pre-incubation periods, Zn²⁺ potentiated then inhibited ATP-responses in a concentration-dependent, but pH-independent, manner.

6. In summary, ATP activity at rP2X₁ receptors was decreased by both extracellular H⁺ and Zn²⁺ and their effects were additive. ATP activity at rP2X₃ receptors was less sensitive to H⁺-inhibition and, in contrast, was potentiated by Zn²⁺ in a pH-independent manner. These differential effects may help distinguish P2X₁ and P2X₃ receptors in whole tissues.

Introduction

Adenosine 5′-triphosphate (ATP) acts as a fast signalling molecule at P2X receptors in central, peripheral and enteric neurons and associated neuro-effector tissues (Burnstock, 1997). Seven P2X receptor subunits (P2X_1–7) have been identified thus far, of which P2X_1–6 subunit mRNA transcripts and receptor proteins are present in neurons (Humphrey et al., 1998; King, 1998). Homomeric P2X₁ and P2X₃ receptors examined in the present study are now classified (Group 1) P2X receptors, which are defined as α,β-meATP-sensitive, rapidly-inactivating, suramin-sensitive (Humphrey et al., 1998). However, there is limited scope to distinguish the operational profiles of P2X₁ and P2X₃ receptors. Previously, we have commented on the effects of H⁺ ions (i.e., extracellular pH, pH_e) and Zn²⁺ ions on ATP-responses at homomeric P2X₂ (Group 2) and P2X₄ (Group 3) receptors, at which significant differences have been observed in their modulatory actions (King et al., 1996; 1997; Wildman et al., 1998; 1999b). We have extended our survey of H⁺ and Zn²⁺ modulation to include Group 1 P2X receptors, in the hope of discriminating between P2X₁ and P2X₃ receptors as well as Group 1 from Groups 2 and 3 P2X receptors.

In an earlier study, the amplitude of ATP-responses at P2X₁ and P2X₃ receptors were shown to be reduced by lowering pH_e, although the H⁺ effect on agonist potency and efficacy was not investigated (Stoop et al., 1997). An inhibitory effect by H⁺ ions was also observed at P2X₄ receptors (Wildman et al., 1999b), which contrasted with a potentiating effect at P2X₂ receptors (King et al., 1996; 1997). Thus, the first objective of this study focused on changes in agonist potency and efficacy at rP2X₁ and rP2X₃ receptors under acidic and alkaline conditions, to see if such changes matched the H⁺ effect at P2X₄ receptors. The second part of the present study focused on Zn²⁺ modulation of Group 1 P2X receptors, and the dependence of such modulatory effects on time and extracellular pH, in the knowledge that this transition metal also affected ATP-activity at P2X₂ and P2X₄ receptors in different ways in a time- and pH-dependent manner (see Wildman et al., 1998; 1999b). Finally, we have compared and contrasted the modulatory activity of H⁺ and Zn²⁺ ions at rP2X₁, rP2X₂, rP2X₃ and rP2X₄ receptors to establish operational profiles distinct for each P2X subunit. Part of this study has been communicated to The Physiological Society (King et al., 1999).

Methods

Oocyte preparation

Xenopus laevis frogs were anaesthetized in Tricaine (0.4% w v⁻¹), killed by decapitation, and ovarian lobes removed surgically. Oocytes (stages V and VI) were defolliculated by a 2-step process involving collagenase treatment (Type IA, 2 mg ml⁻¹ in a Ca²⁺-free Ringer's solution, for 2–3 h) and, thereafter, stripping away the follicular layer with fine forceps. Defolliculated oocytes were stored in Barth's solution (pH 7.5, at 4°C) containing (mM): NaCl 110, KCl 1, NaHCO₃ 2.4, Tris HCl 7.5, Ca(NO₃)₂ 0.33, CaCl₂ 0.41, MgSO₄ 0.82; gentamycin sulphate, 50 μg l⁻¹. Defollicated oocytes were injected cytosolically with either rP2X₁ or rP2X₃ cRNA (40 nl, 1 μg ml⁻¹), incubated for 48 h at 18°C in Barth's solution then kept at 4°C for up to 12 days until used in electrophysiological experiments.

Electrophysiology

ATP-activated membrane currents (I_ATP) (V_h=−60 to −90 mV) were recorded from cRNA-injected oocytes using a twin-electrode voltage-clamp amplifier (Axoclamp 2B). The voltage-recording and current-recording microelectrodes (1–5 MΩ tip resistance) were filled with 3.0 M KCl. Oocytes were superfused with Ringer's solution (5 ml min⁻¹, at 18°C) containing (mM): NaCl 110, KCl 2.5, HEPES 5, BaCl₂ 1.8, adjusted to pH 7.5. Where stated, the pH of the bathing solution was adjusted using either 1.0 N HCl or 1.0 N NaOH to achieve the desired level. Electrophysiological data were stored on a computer using a MP100 WSW interface (Biopac Systems Inc.) and analysed using the software package Acqknowledge III (Biopac).

Solutions

All solutions were nominally Ca²⁺-free to avoid the activation of an endogenous Ca²⁺-dependent Cl⁻ current (I_Cl(Ca)) present in oocytes (Barish, 1983). ATP was prepared in a Ca²⁺-free Ringer's solution (concentrations as stated in the text) and superfused by a gravity-feed continuous flow system which allowed rapid addition and washout. ATP was added for 120 s or until the current reached a peak, then washed out for a period of 20 min. Data were normalized to the maximum current (I_max) evoked by ATP at pH 7.5, including ATP-responses recorded at other pH levels. The concentration required to evoke 50% of the maximum agonist response (EC₅₀) was taken from Hill plots, constructed using the equation log (I/I_max-I), where I is the current evoked by each concentration of ATP.

The effects of extracellular zinc were investigated on agonist activity in two ways. First, C/R curves for the modulatory activity of Zn²⁺ ions were constructed using a submaximal concentration of ATP and data were normalized to the amplitude of control ATP-responses at pH 8.0, 7.5, 6.5 and 5.5. These experiments were carried out for different pre-incubation periods for Zn²⁺ (i.e., 0, 20 and 40 min) prior to the addition of ATP. Second, Zn²⁺ ions were added to ATP solutions and C/R curves for ATP were constructed and normalized to the maximal ATP effect under Zn²⁺-free conditions at pH 7.5. In some experiments using rP2X₃, Zn²⁺ was added to the superfusate 20 min prior to the addition of ATP and C/R curves to ATP constructed.

Statistics

Data are presented as mean±s.e.mean of four sets of data from different oocyte batches. Significant differences were determined by either unpaired Student's t-test or one-way analysis of variance (ANOVA) followed by Dunnett's test, using commercially-available software (Instat v2.05a, GraphPad).

Drugs

All common salts and reagents were AnalaR grade (Aldrich Chemicals, U.K.). Adenosine 5′-triphosphate disodium salt (ATP) and zinc chloride were purchased from Sigma Chemical Co. (Poole, Dorset, U.K.). Drugs were dissolved in Ringer's solution with the pH adjusted to match individual experiments.

Results

Effects of extracellular pH on I_ATP at rP2X₁ and rP2X₃ receptor

Superfused ATP (0.03–30 μM) evoked rapidly-activating, rapidly-inactivating inward currents (I_ATP) in Xenopus oocytes expressing homomeric rP2X₁ receptors. The amplitude of the evoked I_ATP was dependent on extracellular pH (pH_e) and decreased as the superfusate was made more acidic (Figure 1A). This decrease was due to a fall in ATP potency, as judged by a change in the EC₅₀ value (see Table 1) without a concomitant change in the maximum ATP effect (see Figure 2A). Thus, ATP potency was reduced 2 fold at pH 6.5 and 6 fold at pH 5.5, whereas alkaline conditions (pH 8.0) had no effect on agonist potency. The modulatory effects of H⁺ at rP2X₁ receptors were reversed upon readjusting the superfusate to pH 7.5.

Figure 1.

Extracellular pH modulates ATP activity at P2X (Group 1) receptors. (A) Whole-cell inward currents evoked by ATP (1 μM), at four levels of extracellular pH (8.0, 7.5, 6.5, 5.5), at rP2X₁ receptors expressed in Xenopus oocytes. All records from the same oocyte (V_h=−60 mV). (B) Whole-cell inward currents evoked by ATP (3 μM), at the same pH_e levels, at rP2X₃ receptors. All records from the same oocyte (V_h=−90 mV).

Table 1.

ATP potency at Group 1 P2X receptors at different pH_e levels

Figure 2.

pH-dependent changes in ATP potency at P2X (Group 1) receptors. (A) Concentration/response (C/R) curves for whole-cell inward currents (I_ATP) evoked by ATP (10 nM–100 μM), at four levels of pH_e (8.0, 7.5, 6.5, 5.5), at rP2X₁ receptors. The amplitude of I_ATP was normalized to the maximum ATP effect at pH 7.5. (B) C/R curves for I_ATP at rP2X₃ receptors under the same conditions. Data points are means±s.e.mean, n=4.

Superfused ATP (0.1–300 μM) also evoked rapidly-activating, rapidly-inactivating inward currents (I_ATP) from Xenopus oocytes expressing homomeric rP2X₃ receptors. The I_ATP of activated rP2X₃ receptors was insensitive to small changes to pH_e (±1 pH unit), although the amplitude of I_ATP decreased significantly (P<0.05) at pH 5.5 (Figure 1B). This decrease was due to a fall in ATP potency (∼15 fold decrease) (see Table 1), without a concomitant change in the maximum ATP effect (Figure 2B). The modulatory effects of H⁺ at rP2X₃ receptors were also reversed upon readjusting the superfusate to pH 7.5.

Modulatory effects Zn²⁺ ions of I_ATP

Extracellular Zn²⁺ (0.01–300 μM) inhibited I_ATP at rP2X₁ receptors, yet potentiated I_ATP at rP2X₃ receptors, in a reversible manner (Figure 3A, B). Zn²⁺-inhibition of rP2X₁ receptors was not only dependent on concentration but also the pre-incubation time for Zn²⁺ (Figure 4A). Zn²⁺ was approximately 10 fold more potent, as judged by changes in IC₅₀ values (Table 2), when superfused for 20 min or longer prior to ATP application. The ability of Zn²⁺ to inhibit I_ATP at rP2X₁ was not altered appreciably over the range of pH 8.0–5.5 (Figure 4B). Since agonist potency at rP2X₁ is already reduced as the extracellular solution was made more acidic, Zn²⁺ and H⁺-inhibition appear to be additive and to occur by independent processes.

Figure 3.

Zn²⁺ ions modulate ATP activity at P2X (Group 1) receptors. (A) Whole-cell inward currents evoked by ATP (0.3 μM, approx. EC₅₀) before, during and after washout of Zn²⁺ (10 μM; 20 min pre-incubation; at pH 7.5) at rP2X₁ receptors. All records from the same oocyte (V_h=−60 mV). (B) Whole-cell inward currents to ATP (0.3 μM, approx. EC₂₀) before, during and after washout of Zn²⁺ (100 μM; 0 min pre-incubation; at pH 7.5). All records from the same oocyte (V_h=−60 mV). Note that the Zn²⁺ effect at rP2X₁ and rP2X₃ receptors reversed on washout (20 min).

Figure 4.

Zn²⁺-inhibition of ATP-responses at rP2X₁ receptors. (A) Inhibition curves for Zn²⁺ modulation of whole-cell inward currents (I_ATP) evoked by ATP (0.3 μM, approx. EC₅₀), where Zn²⁺ was applied over three pre-incubation periods (0, 20 and 40 min), at pH 7.5. (B) Inhibition curves for Zn²⁺ against I_ATP, at four different levels of pH_e (8.0, 7.5, 6.5, 5.5). Each concentration of Zn²⁺ was applied 20 min prior to and during ATP superfusion. Data points are mean±s.e.mean, n=4.

Table 2.

Inhibitory activity of Zn²⁺ ions at rP2X₁ receptors

Zn²⁺-potentiation of I_ATP at rP2X₃ receptors was dependent on both concentration and the pre-incubation time for Zn²⁺. When applied simultaneously with ATP, Zn²⁺ (0.1–300 μM) potentiated I_ATP by approximately 50% above control responses in a manner described by a sigmoid C/R curve (EC₅₀, 10.9±0.74 μM, n=4) (Figure 5A). However, the effects of Zn²⁺ were more complex when applied prior to ATP, with a 20 min pre-incubation period for Zn²⁺ proving optimal. Under these circumstances, Zn²⁺ showed a greater degree of potentiation, which was then followed by inhibition, in a manner best described by a bell-shaped C/R curve (Figure 5A). The ability of Zn²⁺ to potentiate I_ATP at rP2X₃ receptors (where Zn²⁺ was applied simultaneously with ATP) was not altered appreciably over the range pH 8.0–5.5 (Figure 5B).

Figure 5.

Zn²⁺-modulation of ATP-responses at rP2X₃ receptors. (A) Concentration/response (C/R) curves for Zn²⁺ modulation of whole-cell inward currents (I_ATP) to ATP (0.3 μM, approx. EC₂₀), where Zn²⁺ was applied over three pre-incubation periods (0, 20, 40 min), at pH 7.5. (B) C/R curves for Zn²⁺ against I_ATP, at four different levels of pH_e (8.0, 7.5, 6.5, 5.5). Each concentration of Zn²⁺ was applied simultaneously with ATP. Data points are means ±s.e.mean, n=4.

Effect of Zn²⁺ on ATP potency and efficacy at Group 1 P2X receptors

ATP potency and efficacy at rP2X₁ and rP2X₃ receptors were reassessed in the presence of Zn²⁺ to help explain its modulatory effects. For rP2X₁, Zn²⁺ was applied for 20 min prior to ATP and at a concentration matching its IC₅₀ value (1 μM). Under these conditions, ATP potency was reduced by 5 fold without changing the maximum ATP effect (Figure 6A). EC₅₀ values and Hill coefficients are given in Table 3. For rP2X₃, Zn²⁺ was applied either simultaneously with ATP (0 min) or prior to ATP (20 min) at a concentration (300 μM) which gave maximal potentiation and inhibition for the appropriate pre-incubation period. Zn²⁺-potentiation (0 min pre-incubation) was due to a 3–4 fold increase in ATP potency, without any change in agonist efficacy (Figure 6B). Zn²⁺ inhibition (with 20 min pre-incubation) was due to a 30±5% (n=3) decrease in the maximum ATP effect (decreased agonist efficacy), without any change in agonist potency (Figure 6B). EC₅₀ values and Hill coefficients are given in Table 3.

Figure 6.

Zn²⁺ modulation of agonist potency and efficacy at P2X (Group 1) receptors. (A) concentration/response (C/R) curves for whole-cell inward currents to ATP (30 nM–30 μM) at rP2X₁ receptors, in the absence and present of Zn²⁺ (1 μM), at pH 7.5. Zn²⁺ was applied for 20 min prior to each concentration of ATP. (B) C/R curves for whole-cell inward currents to ATP (3 nM–300 μM) at rP2X₃ receptors, in the absence and presence of Zn²⁺ (300 μM) at pH 7.5. Zn²⁺ was applied either simultaneously with ATP (t=0) or 20 min prior to ATP (t=20). Data points are means±s.e.mean, n=4.

Table 3.

Effect of Zn²⁺ ions on ATP potency at Group 1 P3X receptors

Discussion

H⁺ modulation of P2X (Group 1) receptors

The two members of Group 1 (α,β-meATP-sensitive, fast-inactivating, suramin-sensitive) P2X receptors were affected in a similar manner by acidification of the extracellular solution. Agonist potency was decreased at both rP2X₁ and rP2X₃ receptors, without a concomitant change in agonist efficacy, and this effect was reversed by restoring [H⁺]_o to pH 7.5. Although the H⁺ effect was broadly similar for these two recombinant P2X receptors, there were subtle differences in the way this was manifested. There was a gradual decrease in agonist potency at rP2X₁ receptors over the range of pH 7.5–5.5, and the H⁺ effect was rather modest (a 6 fold increase in the EC₅₀ value for ATP at pH 5.5). In contrast, agonist potency at rP2X₃ receptors was altered only at pH 5.5, at which point the H⁺ effect was more substantial (a 15 fold increase in the EC₅₀ value for ATP).

The H⁺ effect at Group 1 P2X receptors is different from the modulatory activity of H⁺ ions at rP2X₂ (Group 2) and rP2X₄ (Group 3) receptors. H⁺ ions potentiate ATP-responses at rP2X₂ receptors, by increasing agonist potency without changing efficacy, showing maximum activity at levels as low as pH 5.5 (King et al., 1996; 1997, Stoop et al., 1997). However, it was also reported that highly acidic conditions (<pH 5.0) result in a progressive loss in agonist efficacy at rP2X₂ receptors (Stoop & Quayle, 1998). The H⁺ effect at rP2X₄ is characterized by a progressive decrease in agonist potency over the range of pH 7.5–5.5, and a loss of agonist efficacy at pH 5.5 (Wildman et al., 1999b). To some extent, an H⁺ effect has also been observed at rP2X₇ (Group 4) receptors at which acidic conditions (pH 7.5–5.5) progressively inhibited membrane current (through the P2Z ion-channel) and YO-PRO uptake (through the P2Z-activated pore) (Virginio et al., 1997). However, there is no information on changes in agonist potency and efficacy to explain the H⁺-inhibition of ATP-responses at rP2X₇ receptors.

The H⁺ effect is potentially important for P2X receptor signalling for two reasons. First, large pH shifts (2–3 pH units) occur with the localized metabolic acidosis associated with bone fracture (pH 4.7), ischaemia (pH 5.7), inflammation (pH 5.4), epileptic seizures and injuries related to CNS degenerative changes (DeSalles et al., 1987; Chesler, 1990; Ransom & Philbin, 1992; Steen et al., 1992). Second, transient acidic shifts also occur during CNS neurotransmission (Krishtal et al., 1987; Chesler, 1990; Rose & Dietmer, 1995; Yanovsky et al., 1995), a phenomenon due to the way transmitters are packaged in vesicles. Vacuolar H⁺-ATPase (V-ATPase) establishes a H⁺-gradient across the wall of synaptic vesicles, rendering the intravesicular space acidic (pH 5.5) (Cidon & Sihra, 1989). The resultant electrochemical gradient for H⁺ ions assists in concentrating neurotransmitters inside storage vesicles (for a review, see: Moriyama et al., 1992). The acidic shift associated with neurotransmission may have a significant bearing on the sensitization of postjunctional P2X receptors activated by purinergic nerves, particularly during high frequency transmission where significant accretion of vesicular H⁺ will occur at the synapse. Little is known, however, about the pH-dependence of purinergic transmission in smooth muscles where P2X₁ receptors are concentrated. Similarly, the effect of localized acidosis has not been investigated for P2X₃ receptor-mediated currents in sensory nerve endings.

Zn²⁺ modulation of P2X (Group 1) receptors

The Zn²⁺ effect, in contrast to the above H⁺ effect, was dissimilar at rP2X₁ and rP2X₃ receptors–inhibition and potentiation, respectively. The inhibitory action of Zn²⁺ at rP2X₁ receptors was characterized by a fall in agonist potency without any change in agonist efficacy. The immediacy of Zn²⁺-inhibition, where Zn²⁺ was applied simultaneously with ATP, indicated an extracellular locus for its site of action. However, the time-dependency and increased potency of Zn²⁺-inhibition at rP2X₁ receptors also pointed to the involvement of a second intracellular site of action. Time-dependent Zn²⁺-inhibition of ATP-responses was also seen at rP2X₂ receptors (Wildman et al., 1998) and, to some extent, at rP2X₃ receptors (present study).

The actions of Zn²⁺ at rP2X₃ receptors were complex, yet bore a similarity to its actions at rP2X₂ receptors (Wildman et al., 1998). When applied simultaneously with ATP, the main Zn²⁺ effect was potentiation of ATP-responses at rP2X₃ receptors. The same effect occurs at rP2X₂ receptors, although the magnitude of the Zn²⁺ effect is much greater. With Zn²⁺-preincubation, the Zn²⁺ effect at rP2X₃ receptors was characterized by a concentration-dependent potentiation followed by inhibition; the same occurs at rP2X₂ receptors. Potentiation was due to an increase in agonist potency at rP2X₃ receptors, whereas inhibition was primarily due to a decrease in agonist efficacy (by 30%). Zn²⁺-inhibition at rP2X₂ receptors is characterized by a 50% loss of agonist efficacy and a decrease in agonist potency (Wildman et al., 1998). The observed Zn²⁺-inhibition (and Zn²⁺-potentiation) at P2X receptors expressed in oocytes is not due to alterations in ecto-ATPase activity since the enzyme is absent in defolliculated cells (Ziganshin et al., 1995).

The Zn²⁺ effect at P2X (Group 1) receptors is important for two reasons. First, it now provides a simple means to distinguish rP2X₁ receptors from rP2X₃ receptors when, presently, there are no P2X subunit-selective agonists and antagonists. Second, Zn²⁺ ions are taken up and concentrated in synaptic vesicles of defined subsets of central neurons (for a review, see: Smart et al., 1994), although the accumulation of Zn²⁺ in peripheral nerves has not yet been studied. It is not known if vesicular Zn²⁺, like vesicular H⁺, acts as an agent for packaging of neurotransmitters and/or serves as a neuromodulator of receptor function. The Zn²⁺ content of some synaptic boutons is as high as 200–300 μM, and the vesicular concentration may be higher still (Smart et al., 1994). The plasma Zn²⁺ concentration in rat is 21 μM, although much (∼99%) is bound to seral proteins (Walker & Kelleher, 1978). The impact of either vesicular-released or extracellular-fluid Zn²⁺ on purinergic signalling has not been assessed in tissues where P2X₁ and P2X₃ subunits are concentrated.

H⁺ and Zn²⁺ modulation of recombinant rP2X_1–4 receptors

This present study of Group 1 P2X subunits represents a part of an extended survey of H⁺ and Zn²⁺ modulation of P2X subunits known to be associated with neurotransmission. We have already commented at length on the modulatory activity of H⁺ and Zn²⁺ at rP2X₂ and rP2X₄ receptors (King, 1998; King et al., 1996; 1997; Wildman et al., 1998; 1999b). The modulatory activity of H⁺ and Zn²⁺ ions is important because of their presence in synaptic vesicles and likelihood they are co-released with ATP during purinergic transmission. The outcome of the present study, when taken with our past results, shows a spectrum of H⁺ and Zn²⁺ effects at homomeric P2X_1–4 receptors. Table 4 summarizes the available information on these effects and establishes an operational profile for each P2X subunit. Such information on the H⁺ and Zn²⁺ effects, together with distinct agonist profiles for such agents as the naturally-occurring diadenosine polyphosphates series (Brown et al., 1999; Wildman et al., 1999a), may be helpful in the future to identify the presence of P2X subunits in native P2X receptors at a time when P2X subunit-selective antagonists are not available.

Table 4.

H⁺ and Zn²⁺ modulation of ATP-responses at P2X_1–4 receptors

Abbreviations

ATP

adenosine 5′-triphosphate

α,β-meATP

α,β-methylene ATP

cRNA

capped ribonucleic acid

DRG

dorsal root ganglia

EC₅₀

concentration causing 50% of the maximum agonist response

IC₅₀

concentration inhibiting an agonist response by 50%

I_ATP

ATP-activated membrane current

2-MeSATP

2-methylthio-ATP

mRNA

messenger ribonucleic acid

n_H

Hill coefficient

pH_e

extracellular pH

PPADS

pyridoxal-α⁵-phosphate-6-azophenyl-2′,4′-disulphonic acid

TNP-ATP

trinitrophenyl ATP

V_h

holding potential

Zn²⁺

zinc ions