Original Article

pH-dependent Inhibition of Voltage-gated H⁺ Currents in Rat Alveolar Epithelial Cells by Zn²⁺ and Other Divalent Cations

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

	Abstract

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Acknowledgements Appendix References

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 pH_o dependent but were insensitive to pH_i, suggesting that protons and Zn²⁺ compete for external sites on H⁺ channels. The apparent potency of Zn²⁺ in slowing activation was ~10x greater at pH_o 7 than at pH_o 6, and ~100x greater at pH_o 6 than at pH_o 5. The pH_o 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 pK_a 6.2–6.6 and pK_M 6.5, as described by several models. Zn²⁺ effects on the proton chord conductance–voltage (g_H–V) relationship indicated higher affinities, pK_a 7 and pK_M 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 pH_o regulates H⁺ channel gating.

Key Words: metal binding constants, cadmium, pH, hydrogen ion, ion channels

	INTRODUCTION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Acknowledgements Appendix References

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 pH_o and pH_i. 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 (DeCoursey and Cherny 1994 , DeCoursey and Cherny 1995 , DeCoursey and Cherny 1997 , DeCoursey and Cherny 1998 ; Cherny et al. 1995 ). If ion channels are defined narrowly as water-filled pores, then proton channels are not ion channels, although they conduct protons passively down their electrochemical gradient, and independently of other ionic species. In spite of these fundamental differences, it is remarkable how closely proton channels resemble other voltage-gated channels. H⁺ channels activate upon depolarization with a sigmoidal time course, deactivate exponentially, and exhibit a Cole-Moore effect (DeCoursey and Cherny 1994 ) practically indistinguishable from the behavior of delayed rectifier K⁺ channels (Cole and Moore 1960 ). Low pH_o shifts the voltage-activation curves of both H⁺ channels and other ion channels toward positive potentials and slows activation at a given voltage. Voltage-gated proton channels characteristically are inhibited by extracellular polyvalent cations. The transition metals Zn²⁺ and Cd²⁺ have been used most frequently (Thomas and Meech 1982 ; Byerly et al. 1984 ; Barish and Baud 1984 ; Mahaut-Smith 1989 ; DeCoursey 1991 ; Kapus et al. 1993 ; Demaurex et al. 1993 ; DeCoursey and Cherny 1993 ; Humez et al. 1995 ; Gordienko et al. 1996 ; Nordstrom et al. 1995 ), but Cu²⁺, Ni²⁺, Co²⁺, Hg²⁺, Be²⁺, Mn²⁺, Al³⁺, and La³⁺ have similar effects (Thomas and Meech 1982 ; Meech and Thomas 1987 ; Byerly and Suen 1989 ; Bernheim et al. 1993 ; DeCoursey and Cherny 1994 ; Eder et al. 1995 ). To the extent that each has been explored, all of these metal cations shift the voltage dependence of activation (channel opening) to more positive potentials and slow the opening rate (Byerly et al. 1984 ; Barish and Baud 1984 ; Meech and Thomas 1987 ; Mahaut-Smith 1989 ; DeCoursey 1991 ; DeCoursey and Cherny 1993 ; Demaurex et al. 1993 ; Kapus et al. 1993 ; Nordstrom et al. 1995 ; Gordienko et al. 1996 ). These effects resemble those of polyvalent cations on other ion channels (e.g., Frankenhaeuser and Hodgkin 1957 ; Hille 1968 ; Stanfield 1975 ; Gilly and Armstrong 1982 ; Spires and Begenisich 1992 , Spires and Begenisich 1995 ; Arkett et al. 1994 ). Closer examination reveals differences, however. The H⁺ channel is much more sensitive to external ZnCl₂ than are voltage-gated K⁺ channels, which require 1,000-fold higher concentrations to produce comparable effects in squid (Spires and Begenisich 1992 ), 10–100-fold higher concentrations in frog skeletal muscle (Stanfield 1975 ), and 100-fold higher concentrations in Shaker (Spires and Begenisich 1994 ). In addition, the effects of ZnCl₂ on squid axon K⁺ channels are similar for addition to either side of the membrane and internally applied ZnCl₂ is quite potent (Begenisich and Lynch 1974 ). In contrast, we find that ZnCl₂ has qualitatively different effects on H⁺ channels depending on the side of application and thus binds to distinct external and internal sites.

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 pH_o decreases the effectiveness of ZnCl₂. Competition between Zn²⁺ and H⁺ has been noted previously for other channels, including Cl^- (Hutter and Warner 1967 ; Spalding et al. 1990 ; Rychkov et al. 1997 ) and K⁺ (Spires and Begenisich 1992 , Spires and Begenisich 1994 ). We consider whether the pH_o dependence indicates that (a) the active form is not Zn²⁺ but ZnOH⁺, (b) Zn²⁺ and H⁺ compete for the same binding site, or (c) there is noncompetitive inhibition; i.e., protonated channels have a lower affinity for Zn²⁺. We conclude that the external Zn²⁺ receptor is formed by three or more protonation sites, perhaps comprising His residues, that together coordinate one Zn²⁺.

	MATERIALS AND METHODS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Acknowledgements Appendix References

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 (DeCoursey et al. 1988 ; DeCoursey 1990 ), with the exception that we now use elastase without trypsin to dissociate the cells. The rats were anesthetized using sodium pentobarbital. In brief, the lungs were lavaged to remove macrophages, elastase was instilled, and then the tissue was minced and forced through fine gauze. Lectin agglutination and differential adherence further removed contaminating cell types. The preparation at first includes mainly type II alveolar epithelial cells, but after several days in culture the properties of the cells are more like type I cells. H⁺ currents were studied in approximately spherical cells up to several weeks after isolation.

Solutions
Solutions contained 100 mM buffer supplemented with tetramethylammonium (TMA) methanesulfonate (TMAMeSO₃) to bring the osmolarity to ~300 mOsm. One exception was the pH_o 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 (pK_a) (at 20°C) for measurements at the following pH: pH 5.0, Homopipes (homopiperazine-N,N '-bis-2-(ethanesulfonic acid), pK_a 4.61); pH 5.5–6.0 Mes (pK_a 6.15); pH 6.5 BisTris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane, pK_a 6.50); pH 7.0 PIPES (pK_a 6.80); pH 7.5–8.0 HEPES (pK_a 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 (Good et al. 1966 ) were avoided. We could not find information in the literature on the Zn²⁺ or Cd²⁺ binding properties of the buffers used. Therefore, we measured the binding constants for a number of buffers, according to the method described by Good et al. 1966 . This consisted of titrating the buffer alone, and then together with an equimolar amount of the metal salt (usually 10 mmol in a 100-ml vol). The binding constant was calculated from the relationship (Equation 1):

(1)

where K '_M is the metal binding constant, K_a is the proton dissociation constant defined in Scheme 3 (-pK_a 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.

Scheme 3.

View larger version (23K): [in this window] [in a new window]   Figure 1. (A) Effects of ZnCl2 on the H+ current elicited by a 4-s pulse to +10 mV in a cell studied at pH 7.0//5.5. The inset shows the pH of the pipette and bath solutions. (B) The same currents scaled to the same value at the start and end of the 4-s pulse, illustrating the slowing of the activation time course. The steps in the 10 µM record are due to the resolution of the A–D converter.

View larger version (12K): [in this window] [in a new window]   Figure 2. (A) Instantaneous current–voltage relationships in a cell studied at pH 7.0//6.5 before (•) and after () addition of 10 µM ZnCl2 to the bath. A prepulse to +40 mV for control and +100 mV in the presence of ZnCl2 was applied to open H+ channels, followed by a test pulse to the voltage on the abscissae. The current at the start of the test pulse, after the capacitive transient, is plotted. (B) Data from A after correction for the current at the end of the prepulse, and normalized to be equal at +100 mV. Dividing the test current by that at the end of the prepulse corrects for variation in the activation of the gH during different prepulses. The symbols have the same meaning as in A.

View larger version (25K): [in this window] [in a new window]   Figure 3. Effects of divalent metals on the gH-V relationship are incompatible with the idea of voltage-dependent block. (A) The gH-V relationships in a cell studied in the presence of several metals: controls (dashed lines), 0.1 mM CdCl2 (•), 1 mM CdCl2 (), 10 mM CdCl2 (), 10 mM NiCl2 (), and 0.1 mM ZnCl2 (). The sequence was control, all CdCl2 concentrations, control, NiCl2, ZnCl2, and control. (B) The same gH-V relationships in A shifted along the voltage axis so that they superimpose at small gH. Other than small differences in the limiting gH,max, the shape of the voltage dependence appears similar. The voltage shifts applied were: 0, +8, and +34 mV for 0.1, 1.0, and 10 mM CdCl2, respectively, +15 mV for NiCl2 and +18 mV for ZnCl2. (C) The same gH-V relationships plotted on linear axes and scaled to have similar gH,max appear to simply shift along the voltage axis. The scale factor was determined by taking the ratio of gH in the presence of metal to that at +80 mV in the first control measurement. To compensate for the apparent voltage shift (compare A and B), the gH value used for this purpose for the metal data was shifted by 10 mV (1 mM CdCl2, 10 mM NiCl2), 20 mV (ZnCl2), or 30 mV (10 mM CdCl2). All scale factors were <2.5. (D) The steepness of the apparent voltage dependence of "block" by divalent cations is similar to that of the gH-V relationship itself. The data in C are plotted as a ratio of the gH in the presence of metal to that in its absence, at each voltage, using the same symbols as other parts of this figure. There is no block at any voltage at 0.1 mM CdCl2 (•). The control gH-V relationship (C, dashed line) was fitted to a simple Boltzmann distribution and normalized to its fitted maximum. The slope factors of Boltzmann fits were 12.5 mV for control, and for metal ranged from 8 to 13 mV in fits constrained to limit at 1.0.

Good et al. 1966 reported that the affinity of several buffers for Ca²⁺ was generally about five log units weaker than that for Cu²⁺. A notable exception is Mes, which binds Ca²⁺ weakly but Cu²⁺ negligibly (Good et al. 1966 ). We find that Zn²⁺ is bound roughly two log units more weakly than Cu²⁺, consistent with the lower affinity binding of Zn²⁺ than Cu²⁺ to various ionizable groups on proteins (Breslow 1973 ). One exception to this rule is that PIPES did bind Zn²⁺ weakly, whereas Cu²⁺ was bound negligibly (Good et al. 1966 ). All buffers bound Cd²⁺ detectably and to roughly the same extent that they bound Zn²⁺. It should be noted that Table 1 lists log metal dissociation constant (K_M)¹ values, and that a value <1.3 indicates that >50% of the total metal remains unbound. Thus, much of the binding indicated is rather weak and does not preclude using these buffers in studies of metals.

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 (K_sp ~4 x 10^-17; Lide 1995 ). The maximal soluble concentrations: ~40 µM at pH 8, ~4 mM at pH 7, and ~400 mM at pH 6, were not approached during experiments. We encountered solubility problems when titrating the buffers to test for metal binding (above). For this purpose, we usually used 10 mM ZnCl₂, and in fact the solutions began to precipitate just above pH 7. To extend the pH range, buffers with higher pK_a were titrated at 1 instead of 10 mM. The dihydroxide of Cd²⁺ is somewhat more soluble (K_sp 5.27 x 10^-15; Lide 1995 ) than that of Zn²⁺, and the maximal attainable concentration is ~5 mM at pH 8, so solubility was less of a problem. However, when the metal titrations exceeded pH ~8, precipitation commenced.

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 (Cherny et al. 1995 ), the holding potential, V_hold, must be adjusted appropriately. V_hold was set sufficiently negative to the threshold of activation at each pH to avoid Cole-Moore effects (DeCoursey and Cherny 1994 ), but positive enough to avoid unnecessarily large voltage steps.

Conventions
We refer to pH in the format pH_o//pH_i. In the inside-out patch configuration, the solution in the pipette sets pH_o, defined as the pH of the solution bathing the original extracellular surface of the membrane, and the bath solution sets pH_i. 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 (DeCoursey and Cherny 1995 ) (Equation 2):

(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 t_delay 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):

(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.

	RESULTS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Acknowledgements Appendix References

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 current–voltage relationship. The control instantaneous current–voltage (I-V) relationship in Figure 2 A (•) exhibits moderate outward rectification, consistent with previous studies (Byerly et al. 1984 ; Kapus et al. 1993 ; Bernheim et al. 1993 ; Cherny et al. 1995 ; DeCoursey and Cherny 1996 ). The instantaneous I-V relationship in the presence of 10 µM ZnCl₂ () 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 (Frankenhaeuser and Hodgkin 1957 ). In the simplest scenario, the voltage-dependent properties of the channel will simply shift along the voltage axis. Figure 3 A illustrates proton chord conductance (g_H)–V relationships in one cell in the absence (dashed lines) or presence of 100 µM ZnCl₂ (), 10 mM NiCl₂ (), or several concentrations of CdCl₂ (solid symbols). When shifted along the voltage axis, the g_H-V relationships appear quite similar (Figure 3 B), consistent with this mechanism. These metals may reduce the limiting g_H (g_H,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 g_H,max usually became evident, but was difficult to measure accurately. In Figure 3 C, the g_H-V relationships are plotted on linear axes, scaled to the same g_H,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 I_H(ZnCl₂)/I_H(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 8–13 mV. However, if the actual effect is a simple voltage shift of the g_H-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 g_H-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 g_H-V relationship, consistent with a previous study of CdCl₂ on H⁺ currents (Byerly et al. 1984 ). In contrast, ZnCl₂ slows channel opening dramatically, and far beyond its shift of the g_H-V relationship. The effects of ZnCl₂ are dominated by an interaction with the H⁺ channel that results in _act slowing, beyond a simple voltage shift of all parameters.

View larger version (20K): [in this window] [in a new window]   Figure 4. Effects of metals on the activation time constant, act, in the same cell studied at pH 6.0//5.5 as in Figure 3. (A) The voltage dependence of act is plotted in the presence of CdCl2, NiCl2, and ZnCl2 at concentrations indicated in the figure (mM). Four control data sets are plotted as dashed lines. The sequence was control twice, CdCl2, control, NiCl2, ZnCl2, and control. (B) The data in the presence of metals is shifted to more negative voltages, according to the shift of the gH-V relationship observed in this cell (in Figure 3). For clarity, only data at 10 mM CdCl2 (), 10 mM NiCl2 (), and 0.1 mM ZnCl2 () are plotted. Note that the slowing of act by CdCl2 and NiCl2 appears ascribable to a simple voltage shift, whereas ZnCl2 has an additional slowing effect.

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 (Cherny et al. 1995 ). The latter property (_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 g_H-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 pH_o. ZnCl₂ reduces the H⁺ current at each voltage, slows activation, and shifts the voltage dependence of activation to more positive voltages. At each pH_o, the effects are similar, but the concentration of ZnCl₂ required to produce these effects is much greater at low pH_o. In this sense, lowering pH_o 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 pH_o are plotted in Figure 6 and can be thought of as reflecting the "apparent potency" of ZnCl₂ at various pH_o. The concentration required to slow _act twofold is (µM) 0.22 at pH_o 8, 0.46 at pH_o 7, 5.4 at pH_o 6, 89 at pH_o 5.5, and 1,000 at pH_o 5. The apparent potency of ZnCl₂ (estimated for a fourfold slowing of _act where the curves are parallel) decreased only 2.3-fold between pH_o 8 and 7, 10-fold between pH_o 7 and 6, and 103-fold between pH_o 6 and 5.

View larger version (26K): [in this window] [in a new window]   Figure 5. The effects of ZnCl2 are strongly dependent on pHo. Families of voltage-clamp currents are shown at pHo 7.0, 6.0, and 5.0, with pHi 5.5, recorded in the absence (left-most family in each row) and presence of the indicated concentration of ZnCl2. Data in each row are from the same cell, were recorded during an identical family of voltage pulses, and the same calibration bars apply. The cell at pHo 7.0 was held at -60 mV, and pulses were applied from -40 to +20 mV in 10-mV increments. The cell at pHo 6.0 was held at -20 mV and pulses applied from +10 to +70 mV in 10-mV increments. The cell at pHo 5.0 was held at -20 mV and pulses applied from +50 to +100 mV in 10-mV increments.

View larger version (23K): [in this window] [in a new window]   Figure 6. (A) Slowing of act by ZnCl2 depends strongly on pHo (, pHo 8; •, , 7; , , 6.0; , 5.5; , pHo 5) but is independent of pHi. Open symbols indicate cells studied at pHi 6.5, solid symbols at pHi 5.5. Families of H+ currents were recorded in the absence and presence of ZnCl2 in each cell. The H+ currents were fitted by a single exponential after a delay and the act data plotted versus voltage, as illustrated in Figure 4. The ratio of act in the presence of ZnCl2 to that in its absence was measured at several voltages and averaged for each cell. When the voltage range did not overlap (as occurred for only a few cells at high [ZnCl2]), the control value was extrapolated from act data at the highest voltages studied. The mean ratios from three to five different cells at each pHo//pHi are plotted along with SD bars. The dashed line indicates a ratio of 1.0, which means that no effect was observed. (B) The data in A are replotted after correcting for measured metal binding by the buffers used (Table 1). The corrections apply to measurements using PIPES and Mes, for which detectable binding of ZnCl2 was measured. The calculated correction factors that give the fraction of total applied [ZnCl2] that is unbound by buffer are: 0.576 for pHo 7.0, 0.798 for pHo 6.0, and 0.90 for pHo 5.5, calculated from [M]free/[M]total = 1/(1 + K 'M[B-]), where the deprotonated buffer concentration [B-] was calculated by the Henderson-Hasselbalch equation according to the buffer pKa and pH. No correction was applied at pHo 8.0 or 5.0 because no binding of ZnCl2 to HEPES or Homopipes, respectively, was detected (Table 1).

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 pH_o 7 and 8. After correction, the concentration required to slow _act twofold is (µM) 0.22 at pH_o 8, 0.27 at pH_o 7, 4.3 at pH_o 6, 80 at pH_o 5.5, and 1,000 at pH_o 5. The apparent potency of ZnCl₂ (again estimated for a fourfold slowing of _act where the curves are parallel) decreased 1.3-fold between pH_o 8 and 7, 14-fold between pH_o 7 and 6, and 129-fold between pH_o 6 and 5.

Measurements made in the same external solutions with different pipette pH gave no indication that pH_i 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 pH_o in cells studied with pH_i 5.5 (solid symbols and continuous lines) or at pH_i 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 g_H-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 V_rev in each solution (whenever pH_o was changed). Nevertheless, the pH_o sensitivity of the g_H-V relationship to ZnCl₂ (solid symbols) qualitatively resembles that of _act. In fact, the interaction between ZnCl₂ and pH_o manifested in the g_H-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 g_H-V relationship was 0.13 µM at pH_o 8.0, 0.77 µM at pH_o 7.0, 54 µM at pH_o 6.0, 470 µM at pH_o 5.5, and 12.4 mM (by extrapolation) at pH_o 5.0. The apparent potency of ZnCl₂ thus decreased sixfold between pH_o 8 and 7, 70-fold between pH_o 7 and 6, and 230-fold between pH_o 6 and 5. The larger difference between the effective potency of ZnCl₂ between pH 7 and pH_o 8 requires a higher pK_a for the steady state conductance measurement than for the kinetic _act measurement (see DISCUSSION).

View larger version (22K): [in this window] [in a new window]   Figure 7. The shift in the voltage dependence of activation of the gH produced by ZnCl2 or CdCl2 depends strongly on pHo. Mean shift ± SD are plotted for two to seven determinations in each condition (91 total). Filled symbols connected by solid lines represent ZnCl2 and open symbols with dashed lines indicate CdCl2 measurements, and the numbers indicate pHo. The shift was estimated by plotting gH-V relationships and measuring the apparent voltage shift for comparable levels of gH. The estimate was made arbitrarily when gH was large enough to be reliably determined, but always at <50% of gH,max because any reduction in gH,max (which might have a different mechanism) would contaminate the measurement. ZnCl2 and CdCl2 concentrations have been corrected for buffer binding as described in Figure 6. The calculated unbound fraction of CdCl2 was 0.672 at pHo 7 and 0.626 at pHo 6. Because pHi had no detectable effect on externally applied metals (Figure 6), we combined results here for pHi 5.5 and 6.5. For all data at pHo 8.0, pHi was 6.5, measurements at pHo 7 and 6 include both pHi 5.5 and 6.5, and for all data at pHo 5.5 or 5.0, pHi was 5.5.

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 pH_i 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).

View larger version (18K): [in this window] [in a new window]   Figure 8. High concentrations of intracellular ZnCl2 have only subtle effects on H+ currents. The families of H+ currents at pH 6.5//6.5 were recorded in a control cell (A) and with 2.5 mM ZnCl2 in the pipette solution (B), and are scaled according to membrane capacity. The measured Vrev was +4 mV in A and +1 mV in B. Note the slower tail current decay with ZnCl2 in the pipette. Considering binding of ZnCl2 to 1 mM EGTA and 100 mM BisTris in the pipette solution, the free [Zn2+] was ~170 µM. (C) Identical 8-s pulses to +50 mV were applied before and after addition of the pipette solution used in B to the bath, in the same cell shown in A, demonstrating the dramatic effects of this solution applied externally. The threshold for activating outward H+ current shifted from +30 to +60 mV after adding ZnCl2 to the bath (determined using high gain and 5-mV increments). (D) Average (mean ± SEM) H+ current–voltage relationships normalized according to membrane capacity, in 6–10 control cells (•), 9–14 cells studied with 2.5 mM ZnCl2 added to the pipette solution (), and 3 cells studied with 2.5 mM CdCl2 in the pipette solution (), all at pH 6.5//6.5. *Values for ZnCl2 at +80 and +100 mV differ significantly from control (P < 0.05).

Our impression was that there was nothing unusual in the behavior of the g_H in these experiments. H⁺ currents were studied after allowing at least 5–10 min equilibration of the ZnCl₂-containing pipette solution. The amplitude of I_H did not change consistently during the experiment. The mean I_H 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 I_H, 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.

View larger version (12K): [in this window] [in a new window]   Figure 9. Effects of intracellular ZnCl2 on the mean (±SEM) activation time constant, act, and deactivation time constant, tail, in cells studied at pH 6.5//6.5. Tail current decay was fitted with a single exponential in three to nine control cells () and in four to eight cells studied with 2.5 mM ZnCl2 added to the pipette solution (•). All tail values with ZnCl2 in the pipette differ significantly from control (P < 0.01). Values of act obtained by fitting a single exponential after a delay are plotted from 10–12 control cells () and from four to nine cells studied with 2.5 mM ZnCl2 added to the pipette solution ().

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 (Robinson and Stokes 1959 ). V_rev values were consistent with the applied 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, V_rev 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⁺ (Arslan et al. 1985 ). Shortly after addition of 250 µM TPEN to cells studied with ZnCl₂-containing pipette solutions, the tail current kinetics became more rapid. On average, the ratio of _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 pH_o 7.5 or 6.5 (pipette pH) and pH_i 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.

View larger version (19K): [in this window] [in a new window]   Figure 10. Effects of intracellular ZnCl2 on H+ currents in an inside-out patch studied at pH 6.5//6.5. The first family (A) was recorded within 5 min after forming the inside-out patch. The family in B was recorded starting 2.5 min after addition of 2.5 mM ZnCl2, and the family in C was recorded starting 1.5 min after washout. In all parts, the cell was held at -40 mV, and 16-s pulses were applied in 20-mV increments. Calibration bars in A apply to all families.

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 g_H-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 _act–V relationships in Figure 4 according to the shift observed in the g_H-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 pH_o dependent, however. To a first approximation, the pH_o dependence of CdCl₂ was similar to that of ZnCl₂.

Another difference between metals is evident in Figure 7. The shifts of the g_H-V relationships indicate that CdCl₂ is ~30x less potent at either pH_o 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 g_H-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.

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Acknowledgements Appendix References

Polyvalent cations and protons have similar effects on many ion channels (Hille 1968 ; Woodhull 1973 ; Kwan and Kass 1993 ; Arkett et al. 1994 ), perhaps because they bind to similar sites. It has been postulated that the function of voltage-gated proton channels requires at least two distinct types of protonation sites. Conduction likely occurs via a hydrogen-bonded chain (Nagle and Morowitz 1978 ; DeCoursey and Cherny 1994 , DeCoursey and Cherny 1995 , DeCoursey and Cherny 1997 , DeCoursey and Cherny 1998 , DeCoursey and Cherny 1999a , DeCoursey and Cherny 1999b ), in which case the entryway of the "channel" is a protonation site, where H⁺ must bind to initiate permeation. The second type of protonation sites are allosteric regulatory sites (Byerly et al. 1984 ) that govern the strong pH (pH = pH gradient = pH_o - pH_i) dependence of gating; i.e., the 40 mV/U shift in the voltage-activation curve with changes in either pH_o or pH_i (Cherny et al. 1995 ). The pH-dependent gating mechanism was explained economically by assuming identical internally and externally accessible regulatory protonation sites (Cherny et al. 1995 ). More recent evidence suggests the internal and external sites have distinct chemical properties (DeCoursey and Cherny 1997 ).

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 g_H,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 (Cherny et al. 1995 ). Thus, Zn²⁺ (or H⁺) binding at the external site prevents channel opening, and Zn²⁺ (or H⁺) binding at the internal site prevents channel closing.

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 (Byerly et al. 1984 ; Barish and Baud 1984 ; DeCoursey 1991 ; Kapus et al. 1993 ; DeCoursey and Cherny 1993 , DeCoursey and Cherny 1994 ; DeCoursey and Cherny 1996 ; Demaurex et al. 1993 ), whereas others describe the effects as voltage-dependent block (Bernheim et al. 1993 ; Gordienko et al. 1996 ). These views are not equivalent. The voltage dependence of ionic block is generally assumed to arise from the entry of the blocker into the channel pore partway across the membrane potential field, where it gets stuck, physically occluding the pore. Interpreted in terms of voltage-dependent block, metal binding affinity depends strongly on voltage (Bernheim et al. 1993 ; Gordienko et al. 1996 ), whereas effects due to binding to a modulatory site can be explained with a fixed K_M. Because the instantaneous I-V relation was simply scaled down by ZnCl₂ with no detectable voltage dependence (Figure 2), we ruled out the possibility of rapidly reversible binding of Zn²⁺ to a site within the membrane potential field.

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 pK_M = 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 (Eigen and Hammes 1963 ), then the unbinding rate is 9.5 s^-1. Thus, Zn²⁺ probably binds and unbinds in a fraction of a second. If the kinetics are rapid, effects should have been manifested in the instantaneous I-V relation. (b) In normal drug-receptor reactions, the unblock rate is independent of concentration. However, increasing the concentration of ZnCl₂ slowed H⁺ current activation progressively. There was no indication that two populations of gating behavior resulted, as would be predicted if ZnCl₂ modified a fraction of channels that then opened slowly, with the remaining channels opening at the normal rate. A single exponential (after a delay) continued to fit the data at all [ZnCl₂]. Thus it appears that ZnCl₂ binds and unbinds the channel repeatedly during a single pulse, with the slowing effect related to the fraction of time ZnCl₂ is bound to the channel. (c) The steady state voltage dependence of this apparent Zn²⁺ block, defined as the ratio I_H(Zn²⁺)/I_H(control), is quite steep: a simple Boltzmann fit gives slope factors 8–13 mV (Figure 3 D). In terms of traditional voltage-dependent block mechanisms (Woodhull 1973 ), if z is the charge on the blocking ion and 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., Hille and Schwarz 1978 ). Because it is unlikely for a hydrogen-bonded–chain conduction mechanism to support multiple protons simultaneously, especially at physiological pH (DeCoursey and Cherny 1999a ), explaining the high z observed for divalent cation "blockade" is problematic. (d) If ZnCl₂ simply shifted the g_H-V relationship along the voltage axis, then the apparent steepness of the block, defined as the ratio I_H(Zn²⁺)/I_H(control), will be precisely identical to the steepness of the g_H-V relationship in the absence of Zn²⁺. The slopes of the fractional block curves, 8–13 mV (Figure 3D), and control g_H-V relationships, 8–10 mV (DeCoursey and Cherny 1994 ; Cherny et al. 1995 ), are the same, consistent with a simple voltage shift. (e) Finally, any part of the H⁺ channel conductance pathway comprised of hydrogen-bonded chain would not allow Zn²⁺ passage; thus the possibility for voltage-dependent block by Zn²⁺ could exist only in an aqueous vestibule. We conclude that polyvalent cations do not exert their effects by entering into the pore, but instead bind to sites on the channel that are accessible to the solution and outside of the membrane potential field. Binding must be specific because different divalent cations have very different concentration dependencies. For example, effects of micromolar concentrations of Zn²⁺ are seen in the presence of millimolar [Ca²⁺]_o or [Mg²⁺]_o.

View larger version (27K): [in this window] [in a new window]   Figure 11. Comparison of the act data replotted from Figure 6 with the slowing predicted by Equation A6, assuming that the H+ channel cannot open while Zn2+ is bound to its receptor (see text for details). The meaning of the symbols is the same as in Figure 6, and all curves are the predictions for pKa 6.3, pKM 6.5, and cooperativity factor a = 0.03.

Byerly et al. 1984 proposed that divalent cations bind specifically to a site on the external side of H⁺ channels, based on their observation that the g_H-V relationship was shifted less by CdCl₂ than was the _act–V (actually t_1/2-V) relationship. For epithelial H⁺ channels, the disparity in effects on channel opening compared with the g_H-V relationship was even more pronounced for ZnCl₂ than for CdCl₂. A similar sequence of voltage shifts by ZnCl₂ (_act-V > g_H-V > _tail-V) was seen for K⁺ channels (Gilly and Armstrong 1982 ). Their interpretation was that Zn²⁺ binds to a site on the external side of the channel that is exposed to the bath solution only when the channel is closed. This is precisely the nature of the proposed external modulatory protonation site in our model of H⁺ channel gating (Cherny et al. 1995 ). The site must be deprotonated before the channel can open, and during the opening process the site "disappears" and the same site (or a distinct site) appears on the internal side of the membrane. It was necessary to assume that the protonation sites were not accessible to both sides of the membrane at the same time to account for the 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 (Baes and Mesmer 1976 ). The absolute concentration of ZnOH⁺ is a small fraction of the total, and >90% of ZnCl₂ is divalent at pH < 8.0, hence [Zn²⁺] remains relatively constant (Baes and Mesmer 1976 ). Spalding et al. 1990 concluded that ZnOH⁺ was the active form for Cl^-</