Correspondence to: Thomas E. DeCoursey, Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. Fax:312-942-8711 E-mail:tdecours{at}rush.edu.
Released online: 29 November 1999
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Abstract |
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Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl2 reduced the H+ current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H+ current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn2+ were inconsistent with classical voltage-dependent block in which Zn2+ binds within the membrane voltage field. Instead, Zn2+ binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn2+ were strongly pHo dependent but were insensitive to pHi, suggesting that protons and Zn2+ compete for external sites on H+ channels. The apparent potency of Zn2+ in slowing activation was ~10x greater at pHo 7 than at pHo 6, and ~100x greater at pHo 6 than at pHo 5. The pHo dependence suggests that Zn2+, not ZnOH+, is the active species. Evidently, the Zn2+ receptor is formed by multiple groups, protonation of any of which inhibits Zn2+ binding. The external receptor bound H+ and Zn2+ with pKa 6.26.6 and pKM 6.5, as described by several models. Zn2+ effects on the proton chord conductancevoltage (gHV) relationship indicated higher affinities, pKa 7 and pKM 8. CdCl2 had similar effects as ZnCl2 and competed with H+, but had lower affinity. Zn2+ applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H+ currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn2+ receptor may be the same modulatory protonation site(s) at which pHo regulates H+ channel gating.
Key Words: metal binding constants, cadmium, pH, hydrogen ion, ion channels
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INTRODUCTION |
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Voltage-gated proton channels differ from other voltage-gated ion channels, not only in their extreme selectivity for H+, but also in the regulation of their gating by pHo and pHi. The mechanism of permeation is believed to differ radically from traditional ion channels, which comprise water-filled pores through which ions diffuse: proton channels appear to conduct H+ by a Grotthuss-like mechanism of hopping across a hydrogen-bonded chain spanning the membrane (
The effects of metal cations on H+ currents have been characterized variously as voltage-dependent block, voltage shifts induced by electrostatic effects on the voltage sensor, and specific binding to the channel. These interpretations invoke different mechanisms. Voltage-dependent block suggests that the metal ion enters the channel and crosses part of the membrane potential field to reach its block site in the pore. Here we explore the effects of ZnCl2, one of the more potent inhibitors of H+ channels, as a prototypical metal inhibitor. We find that voltage-dependent block is not a viable mechanism. Prominent effects of Zn2+ reflect specific binding that allosterically alters gating.
A key feature of the inhibition of H+ currents by Zn2+ is a profound pH dependence, which has not been described previously. Lowering pHo decreases the effectiveness of ZnCl2. Competition between Zn2+ and H+ has been noted previously for other channels, including Cl- (
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MATERIALS AND METHODS |
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Rat Alveolar Epithelial Cells
Type II alveolar epithelial cells were isolated from adult male Sprague-Dawley rats using enzyme digestion, lectin agglutination, and differential adherence, as described in detail elsewhere (
Solutions
Solutions contained 100 mM buffer supplemented with tetramethylammonium (TMA) methanesulfonate (TMAMeSO3) to bring the osmolarity to ~300 mOsm. One exception was the pHo 7.0 solution made with 70 mM PIPES. External solutions contained 2 mM CaCl2 or 2 mM MgCl2. Internal solutions contained 2 mM MgCl2 and 1 mM EGTA. Solutions were titrated to the desired pH with TMA hydroxide (TMAOH) or methanesulfonic acid (solutions using BisTris as a buffer). A stock solution of TMAMeSO3 was made by neutralizing TMAOH with methanesulfonic acid. TPEN (N,N,N ',N '-tetrakis(2-pyridylmethyl)ethylenediamine) was purchased from Sigma Chemical Co.
Buffers and Their Metal Binding Properties
The following buffers were used near their negative logarithm of the acid dissociation constant (pKa) (at 20°C) for measurements at the following pH: pH 5.0, Homopipes (homopiperazine-N,N '-bis-2-(ethanesulfonic acid), pKa 4.61); pH 5.56.0 Mes (pKa 6.15); pH 6.5 BisTris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane, pKa 6.50); pH 7.0 PIPES (pKa 6.80); pH 7.58.0 HEPES (pKa 7.55). Buffers were purchased from Sigma Chemical Co., except for Homopipes (Research Organics). Buffers such as Tricine and BES that reportedly complex strongly with transition metals (
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(1) |
where K 'M is the metal binding constant, Ka is the proton dissociation constant defined in Scheme 3 (-pKa value), [H+M] is the H+ concentration at the midpoint of the titration curve in the presence of the metal being tested and [B] is the total buffer concentration. The higher the affinity of the buffer for metal, the greater the shift in the titration curve. Table 1 gives the results.
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Solubility of Zn(OH)2 and Other Metal Dihydroxides
An upper limit to the concentration of ZnCl2 is set by the limited solubility of Zn(OH)2 (Ksp ~4 x 10-17;
Electrophysiology
Conventional whole-cell, cell-attached patch, or inside-out patch configurations were used. Inside-out patches were formed by lifting the pipette into the air briefly. Micropipettes were pulled using a Flaming Brown automatic pipette puller (Sutter Instruments, Co.) from EG-6 glass (Garner Glass Co.), coated with Sylgard 184 (Dow Corning Corp.), and heat polished to a tip resistance ranging typically from 3 to 10 M. Electrical contact with the pipette solution was achieved by a thin sintered Ag-AgCl pellet (In Vivo Metric Systems) attached to a Teflon-encased silver wire. A reference electrode made from a Ag-AgCl pellet was connected to the bath through an agar bridge made with Ringer's solution. The current signal from the patch clamp (List Electronik) was recorded and analyzed using a Laboratory Data Acquisition and Display System (Indec Corp.). Seals were formed with Ringer's solution (mM: 160 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4) in the bath, and the zero current potential established after the pipette was in contact with the cell. Bath temperature was controlled by Peltier devices, and monitored by a resistance temperature detector element (Omega Scientific) in the bath.
Because the voltage dependence of H+ channel gating depends strongly on pH, the threshold for activation ranging from -80 to +80 mV at
pH 2.5 and -1.5, respectively (
pH to avoid Cole-Moore effects (
Conventions
We refer to pH in the format pHo//pHi. In the inside-out patch configuration, the solution in the pipette sets pHo, defined as the pH of the solution bathing the original extracellular surface of the membrane, and the bath solution sets pHi. Currents and voltages are presented in the normal sense; that is, upward currents represent current flowing outward through the membrane from the original intracellular surface, and potentials are expressed by defining the original bath solution as 0 mV. Current records are presented without correction for leak current or liquid junction potentials.
Data Analysis
The time constant of H+ current activation, act, was obtained by fitting the current record by eye with a single exponential after a brief delay (
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(2) |
where I0 is the initial amplitude of the current after the voltage step, I is the steady state current amplitude, t is the time after the voltage step, and tdelay is the delay. The H+ current amplitude is (I0 - I
). No other time-dependent conductances were observed consistently under the ionic conditions employed. Tail current time constants,
tail, were fitted to a single exponential (Equation 3):
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(3) |
where I0 is the amplitude of the decaying part of the tail current.
Data are presented as mean ± SD or SEM, as indicated. Significance of differences between groups was calculated by two-tailed student's t test.
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RESULTS |
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Effects of Extracellular ZnCl2 on H+ Currents
The inhibition of H+ currents by external ZnCl2 is illustrated in Figure 1. The H+ current elicited by a pulse to +10 mV is reduced in a concentration-dependent manner by ZnCl2. The rate the current turns on during a depolarizing voltage pulse is slower, as seen more clearly in Figure 1 B, where the currents are scaled to the same value at the end of the pulse. Another effect (explored below) is to shift the voltage dependence of H+ channel gating to more positive voltages. To some extent, the reduced H+ current amplitude and slower activation can be attributed to this voltage shift. One implication is that any attempt to quantitate the apparent "block" of H+ currents by ZnCl2 by measuring the current at the end of a pulse will be arbitrary because the result depends strongly on the length of the pulse and the voltage selected for the measurement. The apparent extent of block at the end of the pulses in Figure 1 would be greatly reduced if longer test pulses were applied and especially if a more positive test potential were selected.
Zn2+ Block Is Not Voltage Dependent
If ZnCl2 binds with rapid kinetics to a site in the H+ channel within the membrane electrical field, this should manifest itself in the instantaneous currentvoltage relationship. The control instantaneous currentvoltage (I-V) relationship in Figure 2 A () exhibits moderate outward rectification, consistent with previous studies () is also plotted. The currents are reduced even though the prepulse was 40 mV more positive. After both sets of currents are scaled to match at +100 mV (Figure 2 B), the currents superimpose, indicating that there is no rapid voltage-dependent block. In some experiments with CdCl2, there was a suggestion that the inward currents were reduced preferentially, but this effect was too small to be sure of, even with data spanning 200 mV. Thus, metals have negligible effects on the instantaneous I-V relation of H+ channels.
The effects of ZnCl2 and other metals might reflect voltage-independent interaction of the metal with the channel or nearby membrane. By binding to or screening negative charges near the external side of the H+ channel, metals could bias the membrane potential sensed by the channel's voltage sensor (), 10 mM NiCl2 (
), or several concentrations of CdCl2 (solid symbols). When shifted along the voltage axis, the gH-V relationships appear quite similar (Figure 3 B), consistent with this mechanism. These metals may reduce the limiting gH (gH,max) slightly, although for the data shown here this effect was smaller than the variability in the control measurements. At higher metal concentrations, some reduction in gH,max usually became evident, but was difficult to measure accurately. In Figure 3 C, the gH-V relationships are plotted on linear axes, scaled to the same gH,max to illustrate their similar shape and slope. The predominant effect is a simple voltage shift.
Even though there is no rapid voltage-dependent block (Figure 2), the apparent voltage shift might conceivably reflect a slow block/unblock process. If we estimate the steady state voltage dependence of this apparent ZnCl2 block in the usual manner by plotting the ratio IH(ZnCl2)/IH(control), the apparent block is quite steep. Figure 3 D shows the ratios for the same experiment as in other parts of this figure. These curves have similar slopes: a simple Boltzmann fit gives slope factors 813 mV. However, if the actual effect is a simple voltage shift of the gH-V relationship, then the apparent steepness of the "voltage-dependent block" will be identical to the steepness of the Boltzmann relationship in the absence of Zn2+. This being the case, the data in Figure 3 D strongly suggest that metals shift the voltage sensed by the channel rather than binding to the channel in a voltage-dependent manner.
ZnCl2 Slows H+ Channel Opening
A prominent effect of ZnCl2 is to slow the activation of H+ currents. We quantified this effect by fitting the turn-on of current during depolarizing pulses to a single exponential, after a delay. This procedure provides a reasonable fit under most conditions. In the presence of ZnCl2, both the delay and act were increased by roughly the same factor. We focussed mainly on metal effects on
act, which are illustrated in Figure 4 for the same cell shown in Figure 3. Because the
act-V relationship is nearly exponential (linear on semi-log axes), it is not possible to distinguish whether
act is slowed or its voltage dependence is shifted, or both. In the simplest case of a Huxley-Frankenhaeuser-Hodgkin voltage shift, all kinetic parameters should be shifted equally along the voltage axis. To explore the extent to which this model might apply, the
act data in Figure 4 B were "corrected" by the voltage shift determined for the gH-V relationship (Figure 3 B). To a rough approximation, the
act effect in CdCl2 and NiCl2 appears to be explainable by this simple voltage shift. Closer examination of Figure 4 B and other data (not shown) at high CdCl2 concentrations indicates that CdCl2 slows activation somewhat more than is accounted for by the shift of the gH-V relationship, consistent with a previous study of CdCl2 on H+ currents (
act slowing, beyond a simple voltage shift of all parameters.
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ZnCl2 and CdCl2 Have Minor Effects on H+ Channel Closing
The tail current decay seemed faster in the presence of external ZnCl2 or CdCl2. However, attempts to evaluate metal effects on H+ channel closing were hampered by the tendency of metals to reduce H+ currents and by the weak voltage dependence of the closing rate (tail changes e-fold in ~50 mV) means that a 35-mV shift of the
tail-V relationship would change
tail at a given voltage by a factor of only two. Examination of data on ZnCl2 and CdCl2 in a number of cells under different conditions gave the impression that the
tail-V relationship may have been shifted in the positive direction at most by roughly the amount that the gH-V relationship was shifted, but little effect was seen in some experiments.
pH Dependence of Metal Effects
Figure 5 illustrates the effects of ZnCl2 on H+ currents at three pHo. ZnCl2 reduces the H+ current at each voltage, slows activation, and shifts the voltage dependence of activation to more positive voltages. At each pHo, the effects are similar, but the concentration of ZnCl2 required to produce these effects is much greater at low pHo. In this sense, lowering pHo decreases the efficacy of ZnCl2. To quantitate the effects of ZnCl2, we measured act and calculated the ratio of
act in the presence of ZnCl2 to that in its absence in the same cell at the same voltage. In most cells, this ratio was the same at all voltages, thus the effect of ZnCl2 is a uniform voltage-independent slowing. Average ratios at several pHo are plotted in Figure 6 and can be thought of as reflecting the "apparent potency" of ZnCl2 at various pHo. The concentration required to slow
act twofold is (µM) 0.22 at pHo 8, 0.46 at pHo 7, 5.4 at pHo 6, 89 at pHo 5.5, and 1,000 at pHo 5. The apparent potency of ZnCl2 (estimated for a fourfold slowing of
act where the curves are parallel) decreased only 2.3-fold between pHo 8 and 7, 10-fold between pHo 7 and 6, and 103-fold between pHo 6 and 5.
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Most of the buffers used bind Zn2+ detectably (Table 1). In Figure 6 B, the data from Figure 6 A are replotted after correcting the metal concentrations for binding by buffer. The correction factors are given in the legend. The main effect is to reduce the shift in apparent potency between pHo 7 and 8. After correction, the concentration required to slow act twofold is (µM) 0.22 at pHo 8, 0.27 at pHo 7, 4.3 at pHo 6, 80 at pHo 5.5, and 1,000 at pHo 5. The apparent potency of ZnCl2 (again estimated for a fourfold slowing of
act where the curves are parallel) decreased 1.3-fold between pHo 8 and 7, 14-fold between pHo 7 and 6, and 129-fold between pHo 6 and 5.
Measurements made in the same external solutions with different pipette pH gave no indication that pHi affects the interaction between externally applied ZnCl2 and act. As illustrated in Figure 6, there was no obvious difference in the effects of ZnCl2 at constant pHo in cells studied with pHi 5.5 (solid symbols and continuous lines) or at pHi 6.5 (open symbols and dashed lines). This result is consistent with externally applied ZnCl2 exerting its effect at the external side of the membrane.
Besides slowing activation, metals also shift channel opening to more positive voltages. This voltage shift was estimated from graphs of the gH-V relationships in the absence or presence of metal and is plotted in Figure 7. This parameter was somewhat arbitrary and less well defined than act, because it required extrapolating the fitted time course of H+ current and measuring Vrev in each solution (whenever pHo was changed). Nevertheless, the pHo sensitivity of the gH-V relationship to ZnCl2 (solid symbols) qualitatively resembles that of
act. In fact, the interaction between ZnCl2 and pHo manifested in the gH-V relationship appears to be somewhat stronger than that for the
act-V relationship. The concentration of ZnCl2 required to produce a 20-mV depolarizing shift of the gH-V relationship was 0.13 µM at pHo 8.0, 0.77 µM at pHo 7.0, 54 µM at pHo 6.0, 470 µM at pHo 5.5, and 12.4 mM (by extrapolation) at pHo 5.0. The apparent potency of ZnCl2 thus decreased sixfold between pHo 8 and 7, 70-fold between pHo 7 and 6, and 230-fold between pHo 6 and 5. The larger difference between the effective potency of ZnCl2 between pH 7 and pHo 8 requires a higher pKa for the steady state conductance measurement than for the kinetic
act measurement (see DISCUSSION).
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Effects of Intracellular ZnCl2 on H+ Currents
Effects of internally applied ZnCl2 were studied in the whole-cell configuration and in inside-out patches. Figure 8 illustrates families of H+ currents in cells studied at pH 6.5//6.5 without (A) and with (B) 2.5 mM ZnCl2 added to the pipette solution. The H+ currents appear generally similar, although closer inspection reveals that the tail currents decayed more slowly in the cell with internal ZnCl2. The pipette solution contained 1 mM EGTA and BisTris buffer (which will bind ~90% of the Zn2+ under these conditions, Table 1), so the addition of 2.5 mM ZnCl2 results in a free [Zn2+] ~170 µM. Figure 8 C illustrates that addition of the pH 6.5 ZnCl2 containing pipette solution to the bath dramatically reduced the H+ current at +50 mV. This result makes it clear that ZnCl2 applied externally is much more effective than when applied internally. Several cells were studied with 2.5 mM ZnCl2 in the pipette at pHi 7.5. HEPES buffer does not bind ZnCl2 detectably (Table 1), hence the free [ZnCl2] was ~1.5 mM. In these cells, the H+ currents also appeared normal (data not shown).
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Our impression was that there was nothing unusual in the behavior of the gH in these experiments. H+ currents were studied after allowing at least 510 min equilibration of the ZnCl2-containing pipette solution. The amplitude of IH did not change consistently during the experiment. The mean IH normalized to the input capacity (Figure 8 D) was reduced significantly (P < 0.05) at +80 and +100 mV in cells studied with 2.5 mM ZnCl2 in the pipette, on average after 29 min in whole-cell configuration. Internal ZnCl2 at high concentrations reduces IH, but this effect is not very pronounced.
Figure 9 illustrates mean act values in cells studied at pH 6.5//6.5 with (
) and without (
) 2.5 mM ZnCl2 in the pipette solution. No difference in the kinetics of H+ current activation was detected. However, channel closing was significantly slower in cells studied with internal ZnCl2. Figure 9 shows mean values of
tail in cells studied with internal ZnCl2 () and in control cells (
). The deactivation rate on average was 3.1-fold slower with internal ZnCl2 (measured between -50 and +10 mV). In three cells studied with 2.5 mM CdCl2 added to the pipette solutions, the average slowing of
tail was 1.8-fold at 10 voltages from -80 to +20 mV (P < 0.05 at each voltage) (not shown). Applied internally, ZnCl2 thus slows closing without affecting activation. In contrast, externally applied ZnCl2 slowed activation and, if anything, accelerated deactivation. Clearly the internal and external sites of action of ZnCl2 are functionally quite different.
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A concern during these experiments was the extent to which ZnCl2 in the pipette solution actually diffused into the cell. ZnCl2 diffusion into the cell will be slowed by binding to cytoplasmic proteins, acting as fixed buffers. That measurable effects on tail were seen is evidence that the ZnCl2 diffused into the cells to a significant extent. The mobility of ZnCl2 is not unusually small (
pH (pH 6.5//6.5, Nernst potential = 0 mV), suggesting that buffer from the pipette solution diffused into the cell. In 11 cells studied with 2.5 mM ZnCl2 in the pipette, Vrev averaged -1.3 ± 2.4 mV (mean ± SEM). To confirm that ZnCl2 entered the cell, we used TPEN, a membrane-permeant metal chelator with a high affinity for Zn2+ (
tail before/after TPEN was 1.65 ± 0.32 (mean ± SD, n = 7), measured at pH 6.5//6.5 at -20 or -40 mV. This is in qualitative agreement with the threefold slowing of
tail observed in the groups of cells studied with or without internal ZnCl2 (Figure 9). Addition of TPEN to three cells studied with metal-free pipette solutions did not affect
tail detectably.
Measurements in Excised Patches
Inside-out patches were studied at pHo 7.5 or 6.5 (pipette pH) and pHi 6.5 (bath pH). Addition of 2.5 mM ZnCl2 to the bath (~170 µM free Zn2+) reduced the H+ current amplitude (Figure 10 B). This effect of ZnCl2 was reversible upon washout (Figure 10 C). The reduction of H+ currents was similar to that observed in whole-cells dialyzed with ZnCl2 containing pipette solutions (Figure 8 D), suggesting that similar concentrations were reached in the whole-cell experiments. There was no clear shift of the voltage dependence of gating. If anything, there was sometimes a small shift to more negative voltages. A small hyperpolarizing shift might be explainable by the slight lowering of pH after addition of ZnCl2 to the solution (0.023 U calculated, 0.05 U measured), due to displacement of protons from buffer. In some inside-out patches, the H+ currents decreased progressively and gradually after addition of ZnCl2. Spontaneous rundown may account for this largely irreversible loss of H+ current. In summary, the inside-out patch data support the conclusion that effects of internally applied ZnCl2 differ qualitatively as well as quantitatively from those of externally applied ZnCl2. Internal application of high concentrations of ZnCl2 produces only modest effects.
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Effects of CdCl2 on H+ Currents
Although we intended to study ZnCl2 as a prototype for the effects of all polyvalent cations on H+ currents, there were subtle differences between the effects of ZnCl2 and CdCl2. Both metals slowed activation and shifted the gH-V relationship to more positive voltages. However, to a first approximation, the effects of CdCl2 could be viewed as a simple shift of all parameters to more positive voltages. "Correction" of the actV relationships in Figure 4 according to the shift observed in the gH-V relationship in Figure 3 normalized the data for CdCl2, but not for ZnCl2. In other words, ZnCl2 has a pronounced additional slowing effect. Examination of
act data in individual cells revealed that ZnCl2 effects could usually be approximated as uniform slowing at all voltages, whereas the relative slowing by CdCl2 sometimes decreased for larger depolarizations. As a result of this subtle difference, there was not a unique "slowing factor" for CdCl2, and we did not try to plot CdCl2 data in Figure 6. The slowing of
act by CdCl2 was strongly pHo dependent, however. To a first approximation, the pHo dependence of CdCl2 was similar to that of ZnCl2.
Another difference between metals is evident in Figure 7. The shifts of the gH-V relationships indicate that CdCl2 is ~30x less potent at either pHo 7 or 6. In contrast, the slowing of act by 100 µM ZnCl2 exceeded that by 10 mM CdCl2 over most voltages (Figure 4 A), and thus there is a >100-fold difference in potency for this effect. Thus the relative potency of the two metals for slowing
act and shifting the gH-V relationship differs. Perhaps distinct binding sites are involved in these effects, and the relative affinities of the metals for the sites differ. ZnCl2 has a high affinity for the site that slows activation, whereas most of the effects of CdCl2 are consistent with binding to a "nonspecific" site that shifts the apparent membrane potential sensed by the H+ channel.
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DISCUSSION |
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Polyvalent cations and protons have similar effects on many ion channels (pH (
pH = pH gradient = pHo - pHi) dependence of gating; i.e., the 40 mV/U shift in the voltage-activation curve with changes in either pHo or pHi (
pH-dependent gating mechanism was explained economically by assuming identical internally and externally accessible regulatory protonation sites (
Given this background, H+ channels might be affected by Zn2+ in several ways. (a) Binding at or near the entry to the channel should inhibit H+ current by preventing H+ binding or reducing the local [H+] available to enter the channel. The attenuation of gH,max at high metal concentrations might reflect local H+ depletion by this mechanism. However, most of the effects of metals are not compatible with metal binding to and occluding the channel entry. (b) Binding to a site remote from the entry but which is sensed by the voltage sensor of the channel could shift the position of the voltage dependency of gating, the most simple mechanism of which would result in all voltage-dependent parameters shifting equally along the voltage axis. This mechanism is consistent with most of the effects of Cd2+ and Ni2+. (c) Binding near the allosteric sites on either side of the membrane might reduce the local [H+] electrostatically, and hence affect gating in the same manner as an increase in pH. The effects of metals are in the wrong direction for this mechanism to apply. (d) Finally, metal binding to the allosteric protonation sites might have a similar effect on gating as protonation of these sites, and might thus mimic the effects of low pH near the site. The details of the effects in this case are hard to predict, because due to differences in binding kinetics and steric factors, Zn2+ can hardly be expected to mimic a single H+, or even two H+. Nevertheless, most of the effects of Zn2+ can be explained by assuming that it binds to the same regulatory sites as protons, and has the same effects as protons in our model (
Zn2+ Is Not a Voltage-dependent Blocker of H+ Channels
Although polyvalent cation effects on H+ currents in various cells are quite similar, some authors have characterized these effects as modification of the voltage dependence of gating (
Even though there is no rapidly reversible block, the more obvious effects of ZnCl2 could be due to a slow time-dependent block/unblock. Five arguments oppose the idea that the slow activation of H+ current in the presence of Zn2+ reflects voltage-dependent unbinding of Zn2+ from the channel. (a) If act in the presence of metals (several seconds) reflects the unblock rate, then block must have very slow kinetics. If we assume that pKM = 6.5 (Figure 11) and that the binding rate of Zn2+ is 3 x 107 M-1 s-1, a characteristic rate of complex formation between Zn2+ and proteins (
is the fraction of the membrane potential sensed by the ion at the block site, then z
2.0, which implies that Zn2+, Cd2+, and Ni2+ traverse
100% of the membrane field to reach the block site. Several examples of
> 1.0 for ionic blockade exist in the K+ channel literature and are traditionally explained by interaction between permeant ions in a multiply occupied channel (e.g.,
observed for divalent cation "blockade" is problematic. (d) If ZnCl2 simply shifted the gH-V relationship along the voltage axis, then the apparent steepness of the block, defined as the ratio IH(Zn2+)/IH(control), will be precisely identical to the steepness of the gH-V relationship in the absence of Zn2+. The slopes of the fractional block curves, 813 mV (Figure 3D), and control gH-V relationships, 810 mV (
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actV (actually t1/2-V) relationship. For epithelial H+ channels, the disparity in effects on channel opening compared with the gH-V relationship was even more pronounced for ZnCl2 than for CdCl2. A similar sequence of voltage shifts by ZnCl2 (
act-V > gH-V >
tail-V) was seen for K+ channels (
pH dependence of gating.
Zn2+ Is the Active Species of Zinc
In solution, zinc exists as several chemical species, whose relative proportions depend strongly on pH. One plausible explanation for the increased apparent potency of ZnCl2 at higher pH is that ZnOH+, rather than the divalent form, is the species acting on H+ channels. As pH is increased, the proportion of ZnCl2 in monohydroxide form, ZnOH+, increases 10-fold/U, up to ~pH 8 (
The pKa sequence generally reveals the proportion of total metal in monohydroxide form at a given pH. If the monohydroxide form were active, then the apparent potency for all of these metals should increase ~10-fold per unit increase in pH, and the pH dependence should saturate around the pKa. We show here that the apparent potency of both ZnCl2 and CdCl2 increase at higher pH, and the pHo dependence saturates for ZnCl2. However, saturation occurs at a pH that is too low by ~1 U, and the change at low pHo is at least 100-fold/U (Figure 5 Figure 6 Figure 7), both inconsistent with the hypothesis that the monohydroxide form is active. If ZnOH+ were the active form, an additional mechanism (e.g., competition with H+) would be required to enhance the pH sensitivity of its effects. Several polyhydroxide forms of zinc (with net negative charge) also are increasingly represented at high pH, but we rule these out as candidates for interaction with the H+ channel because (a) it seems unlikely that anions and protons would compete for the same sites, and (b) the fraction of all of these forms combined at pH 5 is <10-12 of the total ZnCl2 present (
Model of the Interaction between Zn2+ and H+ that Slows Activation
The Appendix explores the predictions of several possible mechanisms of competition between Zn2+ and H+ for hypothetical binding sites on H+ channels. The pHo dependence of Zn2+ effects on act are reasonably compatible with Models 4, 5, or 6 (see Figure 13 for all models). These models assume that the external Zn2+ receptor on proton channels is formed by multiple protonation sites that are accessible to the external solution and that coordinate the binding of a single Zn2+. If H+ and Zn2+ compete directly for the same site(s), then at least two to three protonation sites must exist. If H+ and Zn2+ bind to different sites, then there must be substantial interaction between them, and the range of the pH dependence indicates that protonation of one site lowers the affinity of the remaining site(s) for Zn2+ by a factor ~30. Similar binding constants reproduce the pH dependence of Zn2+ effects using any of several models: pKM is 6.5 and pKa is 6.26.6 and is somewhat model dependent.
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To apply the model equations in the Appendix to real data, it is necessary to define the effect that metal binding has on channel behavior. By making one assumption, we can define the entire body of act data in Figure 6. We assume that when Zn2+ is bound to its receptor on the H+ channel, the channel cannot open. For the simplest case of a two-state channel, with Scheme 1:
where is the opening rate and ß is the closing rate, the time constant is (
+ ß)-1. Because the slowing of
act was voltage independent, ß evidently is negligibly small in the voltage range measured, hence
act
-1. The opening rate will be slowed by the factor (1 - PZn), where PZn is the probability that the receptor is occupied by Zn2+ (the occupancy plotted in Figure 13). Thus the observed time constant will be ~[
(1 - PZn)]-1, and the ratio of
act in the presence of ZnCl2 to that in its absence will be simply (1 - PZn)-1. Given these assumptions,
act is slowed by a factor of 2.0 at the KM of Zn2+.
In Figure 11, the act data from Figure 6 are replotted with smooth curves superimposed that assume Model 6, in which the Zn2+ receptor is formed by three protonation sites and protonation of each site reduces the affinity of the receptor for Zn2+ by a factor a. We selected Model 6 because it comes closest to embodying the pH dependence observed. The entire set of theoretical curves is determined by the assumption that a Zn2+-bound channel cannot open, and by pKM = 6.5, pKa = 6.3, and cooperativity factor a = 0.03. Setting a to 0.03 produces an ~100-fold change in apparent Zn2+ potency between pH 5 and 6 that resembles the
act data. With a = 0.01, the shift was too large, and at a = 0.1 the shift was too small. We could not collect data at pH 4, which might have revealed whether the saturation of the effect at very low pH predicted by this model (Appendix) occurs. The agreement is generally excellent, although the slope of the data appears shallower than that defined by the theory. Expressed in terms of Zn2+ activity rather than concentration, calculated with to the Davies equation (
Model of the Interaction between Zn2+ and H+ that Shifts the gH-V Relationship
The effects of ZnCl2 on the gH-V relationship were modeled in a similar manner as the effects on act. Figure 12 shows the predictions of Model 6 (as in Figure 11), with parameters adjusted to match the ZnCl2 data from Figure 7, which are superimposed. Several differences in the gH-V data compared with the
act data (Figure 11) required different parameters. It was necessary to assume that more than two protonation sites were involved because the shift between pHo 6 and 5 was ~230, whereas 100 is the maximum possible shift for a two-site model. pKM in all equations is defined by the metal concentrationresponse relationship at high pHo, where binding is unaffected by pH. pKa is somewhat model dependent, and is defined by the pHo at which the interaction between metal and H+ saturates. For a given model, this is set by the size of the shift in the high pHo region. Thus, in Figure 12, pKa is 7.0 because this produces a sixfold shift between pHo 8 and 7, as observed in the data. Finally, it was necessary to assume some interaction between binding sites, because pure competition in a three-site model predicts too large a shift at low pHo. The value of the interaction factor, a, is established by the entire shift over the pHo range from 8 to 5. This shift was 105 in the data, and a = 0.01 matched this value. Setting a = 0.02 reduced the range to 3 x 104 and at a = 0 (pure competition) the range was too large, 4 x 105. The mechanistic interpretation is that protonation of one of the sites lowers the affinity of the Zn2+ receptor 100-fold. Assuming the same model, the affinity of Cd2+ for the external metal receptor is lower than that of Zn2+ by ~2 U (roughly pKM 6).
A depolarizing shift in the gH-V relationship at low Popen can be approximated as a reduction of Popen by a constant fraction. Because the slope factor of the gH-V relationship fit by a simple Boltzmann function is ~10 mV (
The gH-V data were best described by pKM = 8.0, pKa 7.0, and a = 0.01 (Figure 12), whereas the optimal values for the same model of act data were pKM = 6.5, pKa 6.3, and a = 0.03 (Figure 11). The different parameter values describing the interactions observed for the gH-V relationship and
act may reflect that the former is a steady state parameter and the latter a kinetic one. Alternatively, distinct metal binding sites may be involved in slowing
act and shifting the gH-V relationship, as suggested by the greater relative potency of ZnCl2 (compared with other metals) for the slowing effect. If so, the "nonspecific" site at which polyvalent metals shift all voltage-dependent parameters simply has a higher pKa than the site that regulates
act. Another possibility is that the Zn2+ receptor has a higher affinity for both protons and Zn2+ when the channel is open. This idea is incompatible with the external Zn2+ receptor being comprised of the regulatory protonation sites that govern gating in our model, because these sites become inaccessible to the external solution when the channel is open (
act and gH-V data simply provides two ways to estimate the binding parameters of the Zn2+ receptor.
Evidence that Metals Bind to the External Site that Regulates pH-dependent Gating
The modeling exercise indicates that protons and polyvalent cations (at least Zn2+ and Cd2+) compete for a common site at the external surface of the H+ channel. Furthermore, the metal receptor can also bind two or more H+, and protonation inhibits metal binding. The best fit was achieved with the assumption that three protonation sites coordinate one Zn2+. We propose that metal binds to the same external modulatory sites at which extracellular protons regulate the gating of H+ channels. Extracellular metals and protons have qualitatively similar effects on channel gating. Both slow activation (increase act), shift the voltage-activation curve (gH-V relation) to more positive potentials, and have relatively small effects on the channel closing rate. In our model (
pH dependence of gating arises from the requirement that three externally accessible sites must be deprotonated for the channel to open. The agreement between the numbers of protonation sites involved in gating and Zn2+ binding may be serendipitous, but lends support to both models.
Internal Metal Binding Site
Although metals produce dramatic effects on H+ currents at quite low concentrations for external application, internally applied ZnCl2 or CdCl2 also altered H+ currents. Deactivation was slowed with no effect on act, and H+ current amplitude was reduced. Because internally applied ZnCl2 had relatively weak effects, we could not study them in as much detail, and could not determine whether Zn2+ and H+ compete for internal sites. Nevertheless, in our model (
Because the effects of internal and external addition were qualitatively different, distinct metal binding sites must exist at the inner and outer surfaces of the channel. In contrast, ZnCl2 has similar effects whether applied externally or internally to K+ channels, on ionic currents (
The Chemical Nature of the Protonation Sites on H+ Channels
To account for the pH dependence of the voltage activation curve of the H+ channel, we originally proposed identical external and internal protonation sites with pKa 8.5 (
-hemolysin channels by introducing His residues (
The much weaker deuterium isotope effect on H+ channel closing than on opening led to the suggestion of Cys as a candidate for the internal regulatory protonation site because sulfhydryl groups typically have smaller pKa shifts in D2O (
Pathophysiological Significance
Because they are more sensitive to polyvalent metal cations than most other ion channels, H+ channels would be among the first to register effects of metal poisoning. Human plasma zinc levels are maintained at ~15 µM (
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Footnotes |
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1 Abbreviations used in this paper: a, cooperativity factor defined in Equation A6; pH, pHo - pHi; gH, proton chord conductance; I-V, currentvoltage; IH, proton current; K'M, metal binding constant; Ka, proton dissociation constant; KM, metal dissociation constant.
Portions of this work were previously published in abstract form (DeCoursey, T.E., and V.V. Cherny. 1999. Biophys. J. 76:A147).
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Acknowledgements |
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The authors are indebted to Tatiana Iastrebova for her meticulous measurements of metal binding affinity to buffers and her technical assistance. The authors appreciate helpful criticism and preprints from Ted Begenisich, John J. Kasianowicz, and Vladislav S. Markin.
This work was supported in part by research grant HL52671 to Dr. DeCoursey from the National Institutes of Health.
Submitted: 4 October 1999
Revised: 8 November 1999
Accepted: 8 November 1999
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Appendix |
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Competition between Zn2+ and H+
If Zn2+ is the sole form of ZnCl2 active on H+ channels, then the strong pHo sensitivity of its effects (Figure 5 Figure 6) could be explained in two ways. Zn2+ and H+ might compete for the same site on the channel, or protonation of the channel might lower its affinity for Zn2+. Several classes of mechanisms encompassing both possibilities can be envisioned. Here we will explore the extent to which the stoichiometry of the apparent competition between metal and protons defines the nature of the metal receptor. To make this analysis as model-independent as possible, we will simply consider the probability that Zn2+ is bound to its receptor. In the DISCUSSION we assumed specific mechanisms by which metals slow H+ channel activation (Figure 11) or shift the gH-V relationship (Figure 12), and determine the model parameters given those assumptions.
If one Zn2+ (M) competes with one H+ (H) for the same binding site or receptor (R), each will have a dissociation constant, defined in Scheme 2 and Scheme 3, respectively (the brackets indicating concentration have been omitted):
and
as KM = kb/kf and Ka = k2/k1.
We define pKM the way pKa is defined, as the negative logarithm of KM. Note that KM as defined here is the inverse of the metal-buffer binding constant defined by
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(A1) |
where the number of free, metal-bound, or protonated receptors is R, RM, or RH, respectively (see
We next consider noncompetitive inhibition, in which Zn2+ and H+ bind independently to different sites, and the effect is observed only when Zn2+ and not H+ is bound. This "noncompetitive inhibition" model is:
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(A2) |
In Equation A2, metal simply becomes ineffective at low pH (Figure 13 B). Furthermore, the "threshold" concentration of metal is the same at all pH. In contrast, the data exhibit large effects of Zn2+ at low pHo, where the concentrationresponse relationship appears simply to shift to higher [Zn2+] (Figure 5 and Figure 6). Furthermore, the threshold concentration that produces detectable effects changes radically with pH. Therefore, we rule out purely noncompetitive models.
The simple competition model could be altered to reflect that Zn2+ and H+ have qualitatively similar effects when bound to H+ channels. Both cations slow activation at a given voltage, and shift the voltage-activation curve to more positive potentials. The formulation of this "both Zn2+ and H+ effective" model is (Equation A3):
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(A3) |
As is apparent from Figure 13 C, if there is competitive binding and Zn2+ and H+ have the same effects, then at low pHo there will be little further effect of adding metal because all the sites are already protonated. There is a small shift of the threshold [Zn2+], but this occurs at the expense of reducing the maximal effect. At least this simple form of the "both effective" class of models is incompatible with the data.
A more complicated model is necessary to explain the ~100-fold shift in apparent potency of Zn2+ between pHo 6 and 5. One possibility is that each channel has multiple protonation sites near enough to each other that two can coordinate a single Zn2+. The divalency of Zn2+ suggests this idea naturally. A channel that coordinates Zn2+ between His and Asp side groups has been described (
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(A4) |
assuming that the two protonation sites are identical and independent. Here Ka = k2/k1, with k1 and k2 defined in the two partial reactions of the degenerate two-step system (
and
Figure 13 D shows the prediction of Equation A4, assuming that pKa = 6.6 and pKM = 6.5. The values of pKa and pKM are established from the pH range at which the pHo sensitivity of Zn2+ diminishes, and from the position of the concentrationresponse curve at high pHo, respectively. In Equation A4, the apparent efficacy of Zn2+ decreases 100-fold/U at low pHo, consistent with the shift observed between pH 6 and 5. The equation describes the act data (Figure 6) fairly well, although the calculated shift between pH 6 and 5 is only 70 rather than 100, because the low pHo behavior is not fully manifest at these relatively high pHo. It might be expected that protonation of the first site would lower the proton affinity of the second site. We modeled this by lowering the pKa of the second protonation reaction, but there were only subtle differences in the predicted behavior for two sites with identical pKa compared with two sites with 1 U different pKa having the same average value.
If the H+ channel were a trimer or tetramer, there might reasonably be three or four equivalent protonation sites. Assuming that protonation of any site prevents metal binding, this purely competitive model is described by (see
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(A5) |
In Equation A5, the shift in apparent Zn2+ potency at low pHo is 103 for n = 3 and 104 for n = 4. Although the data do not exhibit a shift that large, the three-site model 5 (Figure 13 E, "H+ and 1 Zn2+ compete") can simulate the data tolerably well using pKa 6.2 and pKM 6.5 because the 1,000-fold/U shift is attained only at lower pH than the range of the data.
One final variant will be considered, in which the channel has multiple protonation sites that interfere with Zn2+ binding, either allosterically or electrostatically. Protonation of each successive site lowers the affinity of the metal binding site for Zn2+, but protonation and Zn2+ binding are not mutually exclusive. The general "indirect interaction, multimeric site" model is (
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(A6) |
where factor a is a cooperativity factor that is unity for completely independent binding of H+ and Zn2+, decreases as the metal affinity is reduced, and becomes 0 when there is pure competition (in which case Equation A6 reduces to Equation A5). This model behaves as though, in the limit of high and low pH, the affinity of the site for Zn2+ is high or low, respectively. Thus, the shift in apparent metal potency saturates at both high and low pH in contrast with purely competitive Models 1, 4, and 5 (Figure 13), which saturate at high pH only.
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