From the Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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H+ currents in human neutrophils, rat alveolar epithelial cells, and several mammalian phagocyte
cell lines were studied using whole-cell and excised-patch tight-seal voltage clamp techniques at temperatures between 6 and 42°C. Effects of temperature on gating kinetics were distinguished from effects on the H+ current amplitude. The activation and deactivation of H+ currents were both highly temperature sensitive, with a Q10 of 6-9
(activation energy, Ea, 30-38 kcal/mol), greater than for most other ion channels. The similarity of Ea for channel opening and closing suggests that the same step may be rate determining. In addition, when the turn-on of H+
currents with depolarization was fitted by a delay and single exponential, both the delay and the time constant
(
act) had similarly high Q10. These results could be explained if H+ channels were composed of several subunits,
each of which undergoes a single rate-determining gating transition. H+ current gating in all mammalian cells
studied had similarly strong temperature dependences. The H+ conductance increased markedly with temperature, with Q10
2 in whole-cell experiments. In excised patches where depletion would affect the measurement
less, the Q10 was 2.8 at >20°C and 5.3 at <20°C. This temperature sensitivity is much greater than for most other
ion channels and for H+ conduction in aqueous solution, but is in the range reported for H+ transport mechanisms other than channels; e.g., carriers and pumps. Evidently, under the conditions employed, the rate-determining step in H+ permeation occurs not in the diffusional approach but during permeation through the channel itself. The large Ea of permeation intrinsically limits the conductance of this channel, and appears inconsistent
with the channel being a water-filled pore. At physiological temperature, H+ channels provide mammalian cells
with an enormous capacity for proton extrusion.
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INTRODUCTION |
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Voltage-gated H+ channels were described first in snail
neurons (Thomas and Meech, 1982; Byerly et al.,
1984
), and more recently have been found in mammalian cells (DeCoursey, 1991
). A recent burst of interest
in this channel has resulted from its proposed role in
extruding protons from neutrophils and other phagocytes. During the "respiratory burst," NADPH oxidase
secretes superoxide anion to kill bacteria and simultaneously releases protons into the cytoplasm. Henderson and colleagues (1987, 1988a, 1988b) deduced that
electrogenic H+ efflux provided the necessary charge
compensation, on the basis of pH and membrane potential changes in human neutrophils. The presence of
voltage- and pH-activated H+-selective channels in human neutrophils was subsequently confirmed by voltage clamp (DeCoursey and Cherny, 1993
). Despite increasing interest in this channel, its molecular identity
has not been established and numerous questions
about the properties and physiological regulation of
this channel remain unanswered.
Here we explore the effects of temperature on two
fundamental properties of voltage-activated H+ channels: pH-dependent gating and H+ permeation. Interpreting the results requires distinguishing the effects of
temperature on the voltage- and pH-dependent gating mechanism from those on the conductance of the
open channel. A recent suggestion that H+ currents in
murine mast cells have a greater temperature sensitivity than other H+ channels (Kuno et al., 1997) led us to
examine the temperature dependence in several mammalian cells and cell lines. In addition, it is now clear that
the properties of H+ channels differ in different cells.
We studied H+ currents in rat alveolar epithelial cells,
rat macrophages, human neutrophils, human monocyte THP-1 cells, human promyelocyte HL-60 cells, and
mouse microglial BV-2 cells. These cells include both type e (epithelial) and p (phagocyte) H+ channel varieties (DeCoursey, 1998
) and 6 of the 17 mammalian cells
or cell lines and 3 of 5 mammalian species in which H+
currents have been reported. We find similarly high
temperature sensitivity in all mammalian cells.
Voltage-gated H+ channels are extremely selective
for H+ (and deuterium), with no detectable permeability to other cations (Barish and Baud, 1984; Byerly et
al., 1984
; DeCoursey, 1991
; Bernheim et al., 1993
; Kapus et al., 1993
; Demaurex et al., 1993
; DeCoursey and
Cherny, 1994a, 1994b, 1997; Gu and Sackin, 1995
; Gordienko et al., 1996
; Kuno et al., 1997
). Although the
macroscopic conductance increases at lower pHi, the
increase is only 1.7-fold/U decrease in pHi when measured in inside-out patches (DeCoursey and Cherny, 1995
, 1996a
), far less than the 10-fold increase expected if the conductance were proportional to the
permeant ion concentration, [H+]i. In contrast, the H+
conductance of gramicidin (Akeson and Deamer, 1991
;
Cukierman et al., 1997
) and several other H+ permeable channels (reviewed by DeCoursey and Cherny,
1994b
) increases in direct proportion to [H+] up to pH
~0, and then saturates. Thus, the conductance of the voltage-gated H+ channel appears to be nearly saturated at pH 7. Because relatively small changes in gH
are seen when either intracellular or extracellular buffer concentrations were varied 100-fold (DeCoursey
and Cherny, 1996b
), neither buffer diffusion nor direct
proton transfer from buffer to channel can be rate determining steps in conduction. The ratio of H+ to D+
current was 1.9 at 20°C (DeCoursey and Cherny, 1997
),
much larger than 1.41-1.52 for the ratio of H+ to D+
conductivities in bulk solution at 20°C (Lewis and
Doody, 1933
; Roberts and Northey, 1974
). Taken together, these studies suggest that the rate-determining
step in H+ permeation occurs in the pore rather than
in the diffusional approach of either protonated buffer
or H3O+. The activation energy (Ea)1 reported here for
H+ permeation is large enough to rule out conclusively
the possibility that diffusion to the channel is rate determining. That the Ea is as high as for hydrolysis leads
to renewed consideration of this mechanism (Kasianowicz et al., 1987
) as a possible source for a fraction of
the protons that carry current through these channels.
A quintessential feature of all voltage-gated H+ channels is the striking dependence of their voltage-gating
mechanism on pHo and pHi. This interaction was examined systematically in alveolar epithelial cells, where
the voltage-activation curve was found to shift 40
mV/U increase in the pH gradient,
pH = pHo
pHi (Cherny et al., 1995
). The threshold voltage at which the
gH is first detectably activated can be predicted from:
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(1) |
where V0 was typically 20 mV (Cherny et al., 1995), or:
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(2) |
where Vrev is the observed reversal potential (DeCoursey and Cherny, 1997). The importance of the regulation of gating by pH is that the gH is activated only when
there is an outward
pH, thus the channel evidently
functions to extrude H+ from the cell. We have proposed that the regulation of gating by
pH is mediated
by internal and external protonation sites, which are
accessible only to one side of the membrane at a time, and whose accessibility is governed by a conformational
change in the channel molecule that can occur only
when the sites are deprotonated (Cherny et al., 1995
).
The effects of temperature on gating kinetics further
elucidate the gating mechanism. The surprising similarity of the Q10 for activation and deactivation suggests
that the same process is rate determining for both
opening and closing of H+ channels.
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MATERIALS AND METHODS |
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Cells
Human neutrophils.
Neutrophils were isolated from normal human blood by density gradient centrifugation (Schmeichel and
Thomas, 1987), and kept on ice in nominally divalent-free buffer.
Immediately before recording, neutrophils were transferred to
the glass recording chamber and superfused with Ringer's solution (see Solutions, below). In some experiments, fresh blood
from the authors was studied without purification, and neutrophils were identified visually by their size (~8 µm diameter) and
spherical, granular appearance, as described previously (DeCoursey and Cherny, 1993
).
THP-1 cells. THP-1 cells were obtained from American Type Culture Collection (Rockville, MD). Cells were cultured in suspension in RPMI medium supplemented with 0.29 mg/ml glutamine, 10% fetal bovine serum (Gibco Laboratories, Grand Island, NY), 100 U/ml of penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml Fungizone (Amphotericin B; Gibco Laboratories). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Every 2-3 d, about half of the media was replaced with fresh media, and once per week the cells were removed, centrifuged at 1,800 rpm for 10 min at 4°C in an RT6000 refrigerated centrifuge with an H1000B rotor (both from Sorvall, Newtown, CT). The cell pellet was resuspended in fresh media at 1-2 × 106 cells/ml. THP-1 cells are nonadherent. With maintained weak positive pressure, the pipette was placed on or near a cell, and then suction was initiated.
BV-2 cells. BV-2 cells were a gift from Claudia Eder (University of California at Irvine, Irvine, CA). The cells were maintained in DMEM with 10% FCS and 1% L-glutamine.
HL-60 cells. HL-60 cells were obtained from American Type Culture Collection. They were grown in RPMI 1640 media containing 20% FCS. Some cells were studied after treatment with 1% DMSO for 7 d to induce differentiation into granulocytes.
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. Some earlier experiments on cells
isolated with trypsin and elastase are included here. Before invasive procedures were initiated, the rats were anesthetized deeply
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. No changes in the properties of
H+ currents have been observed during this differentiation process. H+ currents were studied in approximately spherical cells
up to several weeks after isolation.
Rat macrophages. Rat macrophages were obtained by lavage during the isolation of type II alveolar epithelial cells. They were studied <1 d after removal from the rat.
Solutions
Most solutions (both external and internal) contained 1 mM EGTA, 2 mM MgCl2, and 100 mM buffer, with tetramethylammonium methanesulfonate (TMAMeSO3) added to bring the osmolarity to ~300 mosM, and titrated to the desired pH with tetramethylammonium hydroxide (TMAOH) or methanesulfonic acid (solutions using bis-Tris as a buffer). The pH 7 and 8 solutions contained 3 mM CaCl2 instead of MgCl2. A stock solution of TMAMeSO3 was made by neutralizing TMAOH with methanesulfonic acid. Buffers (Sigma Chemical Co., St. Louis, MO), which were used near their pK in the following solutions, were: pH 5.5-6.0 Mes; pH 6.5 bis-Tris (bis[2-hydroxyethyl]amino-tris[hydroxymethyl]methane); pH 7.0 Bes (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid); pH 7.5 HEPES; pH 8.0 Tricine (N-tris[hydroxymethyl]methylglycine). In a few experiments (done 5-6 yr ago), TEA+ replaced TMA+, and 20 mM buffer was used. Whether TEA+ is inert with respect to H+ channels is uncertain, but the temperature dependence of the currents appeared consistent with other data using TMA+. In a few other experiments, N-methyl-D-glucamine was used as an impermeant cation instead of TMA+. The initial bath solution was usually Ringer's solution containing (mM): 160 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4.
Electrophysiology
Conventional whole-cell, cell-attached, or excised inside-out
patch configurations were used. Inside-out patches were generally formed by lifting the pipette into the air briefly. Micropipettes were pulled in several stages using a Flaming Brown automatic pipette puller (Sutter Instruments, Co., San Rafael, CA)
from EG-6 glass (Garner Glass Co., Claremont, CA), coated with
Sylgard 184 (Dow Corning Corp., Midland, MI), 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, Healdsburg, CA)
attached to a silver wire covered by a Teflon tube. 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 Electronic, Darmstadt, Germany) was recorded and analyzed using an Indec Laboratory
Data Acquisition and Display System (Indec Corp., Sunnyvale,
CA). Data acquisition and analysis programs were written in
BASIC23 or FORTRAN. Seals were formed with Ringer's solution
in the bath, and the zero current potential established after the
pipette was in contact with the cell. Inside-out patches were
formed by lifting the pipette into the air briefly.
Pulse duration. Pulse duration was adjusted at different temperatures with the intent of balancing two opposing factors. Longer pulses tend to provide a better estimate of both gating kinetics and steady state current amplitudes. However, the longer the pulse, the greater the increase in pHi caused by H+ efflux- mediated depletion of protonated buffer from the cell. Depletion directly distorts the H+ current waveform and also necessitates long interpulse intervals to allow pHi to recover. Most whole-cell measurements above 30-35°C were plagued by signs of changes in bulk pHi due to the massive H+ extrusion during voltage pulses, even though we tried to use relatively small and brief depolarizations to avoid this complication. We generally stopped increasing the temperature when pronounced droop of the H+ current occurred.
Temperature Control
Bath temperature was controlled by Peltier devices in a feedback arrangement, and was monitored by a resistance temperature detector (RTD) element (Omega Scientific, Stamford, CT) placed in the bath near the cell. Bath temperature was recorded at the end of the pulse just before writing to disk, and was stored with each current record. The maximum rate of change of bath temperature was ~0.1°C/s. During temperature changes, the temperature often changed during long pulses. We usually stayed for several minutes at temperatures in intervals of 5-10°C to fix the behavior more accurately. Before lowering the bath temperature, we lifted the cell (via the pipette) because otherwise thermal contraction of the copper housing supporting the bath lifted the chamber enough to smash the pipette tip.
Temperature effects on buffer pKa.
The pKa of most buffers decreases with increased temperature by 0.01-0.02 U/°C. When we
change the temperature, the pKa of the buffers used to establish
the pH of internal and external solutions will change, and consequently H+ will be released or bound by buffer. When the same
buffer, or buffers with similar temperature dependence, are used
in the bath and pipette solutions, temperature will not affect the
pH gradient, pH, but will change the absolute pH. However,
when buffers with different temperature dependences are used
in the bath and pipette solutions,
pH (as well as the absolute
pH) will change. Most experiments were done with Mes or bis-Tris in the pipette solution, both of which have weaker temperature dependence than the most frequently used extracellular
buffers (Bes, less often HEPES, Tricine, or others). Consequently,
pH will decrease at higher temperatures, and EH will
generally change less at higher temperatures and in some situations actually decrease (because changes in
pH and T in the
Nernst equation tend to cancel each other). Over a temperature
range spanning 30°C for the buffers used, the largest change in
absolute pH is 0.42 U (for Tricine), and the largest net change in
pH is ~0.26 U for pH 8.0//6.5 (Tricine//bis-Tris). Most measurements were done at pH 7.0//5.5 (Bes//Mes), where
pH changes 0.04 U/10°C. All solutions are described according to their nominal pH when titrated at room temperature (20-24°C).
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 as 0 mV the original bath solution. 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
delay (as described in DeCoursey and Cherny, 1995
):
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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 (IH)
is defined as (I0
I
). No other time-dependent conductances
were observed consistently under the ionic conditions employed.
The tail current time constant,
tail, was obtained by fitting the
current with a single exponential:
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where I0 is the amplitude of the decaying part of the tail current.
Calculation of Q10 or Arrhenius activation energies. The relative change in a parameter for a 10°C change in temperature, the Q10, was calculated by:
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(3) |
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(4) |
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RESULTS |
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The amplitude and kinetics of voltage-gated H+ currents are strongly temperature dependent. For example, evaporative heat loss from the solution in the recording chamber lowers the temperature by ~1°C, producing a noticeable change in the H+ currents. Fig. 1
illustrates families of H+ currents recorded in a human
neutrophil at 11, 20, and 36°C (note the different time
bases). Increasing the temperature by 25°C increased
the H+ current amplitude >7-fold and the rate of activation (turn on) of the current with depolarization
>20-fold. At all temperatures, H+ currents activated
during depolarizing pulses with a sigmoidal time course,
suggesting that the channel passes through more than one closed state before opening (DeCoursey and Cherny,
1994b; Cherny et al., 1995
). In general, the behavior of
H+ currents appeared to be fairly consistent at all temperatures; that is, after scaling the amplitude and time
scales, there were no obvious changes in the characteristic properties or appearance of the current waveforms.
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Fig. 2 illustrates tail currents and Vrev measurements
in the same neutrophil as in Fig. 1. A depolarizing
prepulse opened many H+ channels, and then the
membrane was repolarized to various potentials. The
time course of current decay reflects the progressive
closing of channels at each potential. The tail current
decay was well fitted by a single exponential, giving tail,
the time constant. The rate of channel closing (1/
tail)
is evidently highly temperature sensitive (note the
change in time base), as was the rate of channel opening in Fig. 1. When
tail was measured over a wide voltage range at different temperatures (Fig. 2 C), the
tail-V relationship appeared to scale uniformly at all potentials.
Our model (Cherny et al., 1995
) predicts a 5% steeper
slope of the
tail-V relationship at higher temperatures
(V.S. Markin, personal communication). However, the
measurement is not sufficiently accurate to detect this
subtle a change. To a first approximation, the Q10 of
tail is independent of voltage.
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The reversal potential Vrev was determined from tail
current measurements like those in Fig. 2, A and B. In
the illustrated neutrophil, Vrev was close to 80 mV at
both 20.5 and at 36.5°C. The Nernst potential for H+,
EH, at nominally pHo 7.0//5.5, is
87 mV at 20.5°C and
88 mV at 36.5°C after correction for shifts in the true
pH due to the temperature dependence of the pKa of
the buffers used (see MATERIALS AND METHODS). In
other cells, Vrev generally was unchanged or somewhat
more positive at lower temperatures. The actual value obtained for Vrev is sensitive to depletion of protonated
buffer from the cell by previous pulses, including the
prepulse used in standard tail current measurements.
Within the reliability of the measurement, temperature
does not appear to alter Vrev beyond the small change
in EH predicted by the Nernst equation.
The steady state voltage dependence of the gH (the
activation curve) was generally similar at all temperatures. This result is important partially for technical
reasons. The comparison of H+ current kinetics at a
fixed test potential at different temperatures would become less valid if the Popen were different. A hyperpolarizing shift in Popen at high temperature would tend to
artificially enhance the temperature dependence of the
activation time constant, act, and perhaps reduce the
temperature dependence of
tail. The voltage dependence of channel opening is not easy to evaluate quantitatively because the chord conductance (gH) often
does not saturate, the gH-V relationship is poorly described by a Boltzmann function, whole-cell currents
are susceptible to depletion effects, and for other reasons discussed at more length elsewhere (DeCoursey, 1991
; DeCoursey and Cherny, 1994a
, 1994b
; Cherny et
al., 1995
). Therefore, several approaches were taken
(not all are illustrated). In Fig. 3 A, gH was determined
in an inside-out patch from an alveolar epithelial cell,
from the amplitude of an exponential fit to the rising
phase of H+ currents. Using the fitted amplitude corrects data in which activation did not achieve steady
state during the pulse. In this experiment, activation
appeared to occur at more negative potentials (by
~10-15 mV) at higher temperatures. However, both
the tremendous increase in gating kinetics, as well as
the increase in H+ current amplitude, will tend to give
this impression, even if there were no true shift. Small
changes in
pH and EH due to temperature effects on
buffers predict net shifts of a few millivolts in the depolarizing direction at higher temperatures (see Fig. 3,
legend). We also looked for changes in Vthreshold, the
threshold potential at which time-dependent outward
current was first detectable. In some experiments, careful examination revealed little or no shift, whereas in
other experiments shifts of 5-10 mV occurred, with activation usually occurring at more negative voltages at higher temperatures. The impression gained from these
attempts was that any shift in the voltage-activation
curve was smaller than could be demonstrated convincingly. We cannot distinguish whether increasing the
temperature produced a small (~10 mV) hyperpolarizing shift or no effect. Likewise, we cannot resolve
whether high temperature might have steepened the
gH-V relationship somewhat as predicted by our model
(Cherny et al., 1995
).
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The time course of H+ currents, at least for moderate
depolarizing pulses, was reasonably well described by a
single exponential rise after a delay. Fig. 3 B illustrates the
voltage dependence of the activation time constant, act,
measured at several temperatures in the inside-out
patch shown in Fig. 3 A. As was found for
tail, the Q10
appeared to be independent of the voltage at which the measurement was made. Our model (Cherny et al., 1995
)
predicts some temperature dependence of the slope of
the
act-V relationship. However, within the accuracy of
the measurement, both kinetic parameters appeared
simply to scale uniformly with temperature at all voltages.
Similar results were obtained in whole-cell experiments.
To evaluate H+ channel gating and conductance
over a wide range of temperature, we used a moderate
depolarizing test pulse, followed by repolarization either to Vhold or to another voltage at which the tail current
decay was resolvable. This pulse protocol permitted obtaining all four parameters nearly simultaneously: act,
the delay time,
tail, and IH. As the temperature was varied, the pulse durations were adjusted to resolve the kinetic parameters. Low temperatures required very long
pulses (up to 80 s), with the result that the temperature
sometimes changed significantly during the pulse.
Some hysteresis in the data is to be expected in this situation. Fig. 4 illustrates the temperature dependence of the parameters studied in a rat alveolar epithelial
cell. All four parameters have roughly linear temperature dependence (in a semi-log plot), and thus each
can be described by a single Q10. The same data are
plotted in Fig. 4 B in a conventional Arrhenius plot in
which the slope gives the Ea.
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Comparison among Different Cells and Mammalian Species
Fig. 5 illustrates the temperature dependence of the
four parameters studied in several types of cells. Several
features are immediately apparent. First, the three kinetic parameters, act, delay, and
tail are all highly temperature sensitive. A second result that is somewhat
unusual in temperature studies on ion channels is that
there are no obvious changes in the slopes of these
curves. The data were well described by a straight line on the graphs, giving a single Q10 value over the entire
temperature range. Q10 values from experiments like
these are summarized in Table I. The third result evident in both Fig. 5 and Table I is that the three kinetic parameters have nearly the same Q10. The similarity of Q10 values for the three kinetic parameters explains why the general appearance of the currents
appeared to simply scale with temperature (compare
Figs. 1 and 2).
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The H+ current amplitude increased substantially
with temperature. However, its measurement was complicated by several factors. At low temperature, activation was very slow, and the pulses were sometimes not
long enough to reach steady state. We extrapolated the
fitted exponential rise to obtain IH. At high temperatures, the currents were quite large and we sometimes
observed decay of outward currents above ~30°C. H+
channels do not inactivate (DeCoursey and Cherny,
1994b), and this decay is almost certainly the result of
increased pHi due to the large efflux of H+ comprising
current flow (see DISCUSSION). Other factors that could
artifactually decrease IH at high temperature include
spontaneous clogging of the pipette tip and movement
of the cell relative to the pipette due to thermal expansion of the copper support for the chamber (used to
transfer heat to the preparation). Because many of
these complications can be avoided or minimized using the inside-out patch configuration, we considered this
approach to provide the most reliable estimate of the
temperature dependence of IH.
Inside-Out Patch Experiments
Several measurements were made in inside-out patches
excised from rat alveolar epithelial cells. Although the
smaller current amplitudes compared with whole-cell
measurements made the extraction of kinetic parameters less precise, the problems associated with pHi
changes due to depletion of protonated buffer by H+
currents are greatly reduced. Fig. 6 illustrates families
of H+ currents at several temperatures in an inside-out
patch. The parameters that could be extracted are plotted in Fig. 5 D, as was done for whole cell data. At
higher temperatures, the time-independent "leak" current appears to increase. Because the IH amplitude is obtained by fitting the current to a delay plus a single
exponential, it reflects only the time-dependent component of outward current. If some part of the initial
jump in outward current at higher temperature reflects
H+ current, for example if the decay of capacity current obscures the initial rise in IH at higher temperatures where activation becomes rapid, then the Q10 of
IH will be underestimated by this procedure. We could
not resolve tail currents well enough to estimate tail reliably over a large temperature range in most patches. The values of
act, summarized in Table I, are generally
similar to those from whole-cell experiments. Notably
different is IH. The H+ current amplitude in patches
continued to increase at higher temperatures (instead
of saturating above ~30°C). The Q10 extracted from
patches even in the higher temperature range (>20°C,
Table I) was larger than for whole-cell experiments. Because the use of excised patches minimizes depletion
problems, we believe that these data are more reliable
than those obtained in whole-cell experiments.
|
The IH data from patches exhibited clear nonlinearity of the type observed to a lesser extent in whole cell IH data. In Fig. 7, IH data from three patches with different current amplitudes are plotted. The Q10 is 2-3 at 20-30°C, but much larger (4-6) at temperatures below 20°C. The curvature is in the direction that would be attenuated in an Arrhenius plot; however, the Arrhenius plots in Fig. 7 B are still clearly nonlinear, with Ea ~15 kcal/mol at high temperatures and ~30 kcal/mol at low temperatures. Activation energies clearly vary depending on the temperature range studied. In Tables I we list values from above and below 20°C.
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DISCUSSION |
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The main results are: (a) the rate of activation of the gH
is highly temperature sensitive. The Q10 for both the delay and act was about the same, ranging from 6 to 9 in
various cells. (b) The rate of deactivation also has a
high Q10 of 6-8, practically identical to that for activation. (c) There were no obvious break points in the
temperature dependence of the three kinetic parameters studied. (d) The Q10 of the H+ conductance is
high, >2 in whole-cell measurements, and 2.8 in inside-out patches >20°C and 5.3 at <20°C. (e) The Arrhenius plot of IH is nonlinear, being steeper at low temperature. (f) The temperature dependence of both H+
currents and H+ channel gating kinetics is quite similar
in different mammalian cells.
H+ Channel Gating Is Steeply Temperature Dependent
Table I summarizes the temperature dependence of
three kinetic parameters reflecting channel opening
(act and delay) and closing (
tail). For all cells studied,
the mean Q10 values were similar, both from one cell
type to another, and, more remarkably, for all three kinetic parameters. For other ion channels, opening and
closing (
act and
tail) do not as a rule have the same Q10 (Table II), and in most cases the Q10's of inactivation
(or block) and recovery appear to be radically different. The Q10 values in Table IA for H+ currents range
from 6 to 9, which inspection of Table II shows is at the
upper end of reported values for gating of various ion channels. Most channel gating processes have Q10 values near 3, generally taken to indicate significant conformational rearrangement of the channel molecule. A
high Q10 for K+ channel inactivation was ascribed to interaction between two peptide moieties, at least one of
which evidently has a low probability of adopting the
correct conformation to permit binding (Murrell- Lagnado and Aldrich, 1993). Alamethicin pore formation (listed as
act in Table II) has a Q10 of 9 at low temperatures. This result is intriguing because alamethicin
is believed to form channels in which 6-10 molecules
assemble in the membrane in a barrel-stave arrangement (Boheim and Kolb, 1978
), reminiscent of one of
the hypothetical physical depictions of our gating
model, in which several channel protomers assemble in
the membrane to form a functional channel (Cherny
et al., 1995
). Another intriguing parallel is the high Q10
of a slow gating process in a Cl
channel that, like the
H+ channel, is gated by its permeant ion, interpreted as
reflecting channel subunit interaction (Pusch et al.,
1997
). In general, the high Q10 for H+ channel gating is
compatible with substantial conformational changes in
the channel.
|
|
We proposed a model of H+ channel gating in which
opening requires deprotonation of an externally accessible site(s), followed by a conformational change that
shifts the accessibility of the protonation site(s) to the
internal side, and finally stabilization of the open configuration by protonation from the inside (Cherny et
al., 1995). This model illustrated how gating could be
regulated by voltage and the pH gradient,
pH (pHo
pHi). Although the model is hypothetical, it is difficult
to conceive of a mechanism that does not incorporate
the general ideas of regulatory protonation sites whose
accessibility switches from one side of the membrane to
the other. The voltage dependence of gating in our
model could arise either from voltage-dependent proton binding to sites inside "proton wells" analogous to
those in H+ ATPases (Mitchell and Moyle, 1974
) or
from a voltage-dependent conformational change, as is
more typical of voltage-gated ion channels. The protonation/deprotonation reactions at the internal site are
rate determining, with the conformational change being rapid and in quasi-equilibrium. Because deuterium
slowed activation threefold with only minor effects on
deactivation, we suggested that a voltage-dependent
closing step might precede the internal deprotonation
step (DeCoursey and Cherny, 1997
).
In this context, the similarity of Q10 values for act, delay, and
tail was surprising. Although we cannot rule
out the possibility that several processes could have the
same high Ea, the simplest explanation is that the same
energy barrier is rate determining for all three parameters. The energy wells on either side of the barrier must
be relatively symmetrical. The Ea for this process is evidently 30-38 kcal/mol, which is substantially larger
than any of the proton-related processes listed in Table
III. Specifically, the Ea is much larger than 7 kcal/mol
for ionization of the imidazole group of histidine
(Reeves, 1977
), a candidate for the external regulatory
protonation site, proposed on the basis of deuterium isotope effects on gating (DeCoursey and Cherny,
1997
). In a linear gating scheme, the delay presumably
reflects early events in the opening process,
act reflects
the entire gating sequence, and
tail must reflect the final transition between the open state and a neighboring closed state. It appears paradoxical that the same
process could determine the relatively rapid tail current decay and the slower activation. A possible explanation for the similarity of Ea for all three kinetic parameters is that each of several channel subunits must
undergo an identical or similar complex first-order
conformational change during opening, but a reverse
transition in only one subunit is sufficient to close the
channel. This is essentially the Hodgkin-Huxley (1952)
model for Na+ and K+ channel gating. This hypothesis
would also explain the surprising similarity of Ea for the
delay,
act, and
tail. In a multiple independent subunit
channel, the ratio of delay to
act is fixed (R.W. Aldrich
and F.T. Horrigan, personal communication).
|
The temperature dependence of gating was nearly linear in
Arrhenius plots.
All three of the kinetic parameters measured had roughly linear temperature dependence
over the range 6-42°C. For a constant Arrhenius activation energy, Ea, the temperature dependence in simple
semi-logarithmic plots (e.g., Figs. 4 A, 5, and 7 A) is slightly nonlinear, becoming steeper at low temperatures. Our data do not resolve the fine distinction between linearity and the predicted Arrhenius curvature,
and appeared linear on both types of plots (compare
Fig. 4, A and B). Many studies of other ion channels describe break points in conductance (Lass and Fischbach, 1976; Chiu et al., 1979
; Hagiwara and Yoshii,
1980
; Quartararo and Barry, 1988
) or gating kinetics
(Schwarz, 1979
; Chiu et al., 1979
; Kirsch and Sykes,
1987
), which occur at 4-20°C. Others report no clear
break points, although the Q10 increases at lower temperatures (Boheim and Kolb, 1978
; Kimura and Meves,
1979
; Kukita, 1982
; Beam and Donaldson, 1983
; Pahapill and Schlichter, 1990
). Breakpoints in Arrhenius
plots are generally taken to indicate phase transitions of membrane lipids. Krasne et al. (1971)
reported that
"freezing" artificial bilayer membranes abolished the
conductance mediated by the carriers nonactin and
valinomycin, but had little effect on the conductance of
the gramicidin channel. Here we saw no consistent or
reliable evidence of a break point in the temperature
dependence of any kinetic parameter. The absence of a
break point could mean that H+ channel gating is relatively insensitive to the fluidity of the surrounding
membrane (see Beam and Donaldson, 1983
). A more
likely explanation is that the membranes of the cells
studied do not exhibit sharp phase transitions in the
temperature range studied. The linearity of the Arrhenius plots suggests that the same process is rate determining over the entire temperature range.
Arrhenius plots of IH are nonlinear.
In contrast to the
kinetic parameters, IH usually changed more steeply at
low temperatures, and tended to saturate at high temperature (>30°C). In cells in which IH saturated, the
currents often decayed during the pulse (not shown).
H+ channels do not inactivate, and this decay is almost
certainly the result of increased pHi due to the large efflux of H+ during current flow. Large IH decreases the
driving force by increasing pHi, as has been demonstrated by changes in Vrev (DeCoursey, 1991; DeCoursey
and Cherny, 1994b
), or deduced from pHi measured by
microelectrodes (Thomas and Meech, 1982
; Meech
and Thomas, 1987
) or fluorometric dyes (Kapus et al.,
1993
). During even moderate depolarizations at high
temperature, there was sometimes evidence of depletion. Our interpretation is that high temperature exacerbates the depletion of protonated buffer from the
cell (and consequent increase in pHi), in spite of the
high (100 mM) concentration of buffer in all solutions.
Thus, the apparent saturation of IH at high temperature is not ascribable to events occurring near the channel. Higher Q10 values were obtained for IH measured
in inside-out patches than in whole-cell experiments,
whereas Q10's for gating kinetics were similar. The most
likely explanation is that depletion of protonated buffer
due to H+ efflux is less problematic in excised patches
because there are much smaller diffusion barriers between either side of the membrane and an effectively
infinite volume of buffered solution.
IH Has Abnormally Strong Temperature Dependence
The average Q10 for IH in whole-cell measurements was
2.1-3.1 in various mammalian cells, and was higher in
patches (Table IA). For reasons discussed above, we
consider Q10 values from inside-out patches to be more
reliable for IH (but less reliable for the kinetic parameters). The Q10 in patches averaged ~2.8 at >20°C and
increased to 5.3 at <20°C (Table IA). As discussed in
the context of Fig. 6, overestimation of the leak current
would tend to decrease the apparent Q10. Temperature
effects on the pKa of buffers will affect these values by
changing both the absolute pH and pH. The decrease
in pHi at higher temperature will tend to increase IH,
presumably by ~1.7-fold/U (DeCoursey and Cherny, 1995
, 1996a
). Most of the measurements were made
with Bes externally and Mes internally, for which
pH
will decrease at higher temperatures, essentially offsetting the increase in RT/F. Correcting for the decrease
in pHi at higher temperature lowers the Q10 values to 2.0-2.9 in whole-cell and 2.6-5.0 in excised patch measurements. These values greatly exceed practically all
values reported for ion permeation through other channels (Table II), and thus require some explanation.
An increase in IH with temperature could reflect increased single-channel conductance or open probability, Popen. If the opening and closing rates had different
temperature dependence, Popen would vary with temperature. Three observations indicate that Popen does
not change with temperature. First, the gH-V relationship was not convincingly shifted. Second, the kinetic
parameters of gating (delay, act, and
tail) and the IH
waveform in general all appeared to simply scale uniformly with temperature. The temperature dependence of neither
act nor
tail was detectably voltage dependent. Finally, the H+ current variance at 20°C increases sharply with depolarization, and then plateaus
or decreases with further depolarization (V.V. Cherny
and T.E. DeCoursey, unpublished observations), suggesting that for large depolarizations Popen > 0.8 at
20°C, which would severely limit any possible increase
at higher temperatures by this mechanism. None of the
arguments is conclusive, and none rules out mechanisms in which the number of functional channels changes with temperature. Insertion of channels at
high temperature by vesicle fusion can be ruled out because lowering the temperature rapidly reverses the effects of temperature on IH. Finally, we cannot eliminate
the possibility that a rapid gating process (i.e., "flicker")
that is perhaps not in the normal opening pathway
might alter the effective unitary current with some arbitrary temperature dependence. In the discussion that
follows, we assume that the temperature dependence
of IH reflects changes in unitary conductance.
The H+ current is not limited by bulk diffusion.
Limitations
to channel permeation can occur at several stages, as
delineated for gramicidin by Andersen (1983). The
rate-limiting step could occur during diffusion to the
mouth of the channel, entry into the channel, permeation through the pore, the exit step, or diffusion away
on the distal side. For H+ channels, additional possible
rate-limiting steps include buffer diffusion, protonation/deprotonation reactions, and hydrolysis. Because
varying the external or internal buffer concentrations from 1 to 100 mM changed IH less than twofold (DeCoursey and Cherny, 1996b
), neither diffusion nor protonation/deprotonation of buffer limits IH. The present
results appear to rule out the possibility that diffusion
of free H+ is rate determining. This conclusion is consistent with the larger deuterium isotope effect on H+
channel currents than on conduction in bulk solution
(DeCoursey and Cherny, 1997
).
Temperature dependence of H+ conduction in water.
The
electrical conductivity (ionic mobility) of H+ is anomalously higher than that of any other cation, by a factor
of ~5 (Eigen and DeMaeyer, 1958; Robinson and
Stokes, 1959
). This observation is explained by the
unique conduction mechanism for H+. The proton in
aqueous solution exists mainly (96-99% of the time) in
association with a particular water molecule as a hydronium ion, H3O+ (Conway et al., 1956
). Any of the three
protons may leave this molecule to conduct current.
Protons thus hop from one water molecule to the next
(de Grotthuss, 1806
) by a mechanism referred to as Grotthuss, water wire, or prototropic transfer (Lengyel
and Conway, 1983
). The activation energy for H+ conduction is lower than for other ions, and decreases rapidly with increasing temperature. The Q10 for H+ conductivity,
0, decreases from 1.2 between 5 and 15°C to
1.11 between 35 and 45°C (Landolt-Börnstein, 1960).
The conductivity of H+ traditionally has been separated
into ordinary hydrodynamic conduction (that expected
if H3O+ diffused as an invariant molecular species like
other ions) and "excess" prototropic conductivity of
H+; e.g.,
H+
Na+ (Hückel, 1928
). Hydrogen bonds
between molecules facilitate H+ conduction by the
Grotthuss mechanism. The excess prototropic conduction decreases strongly with increased temperature:
(
HCl
KCl)/
KCl is 2.26 at 273°K and 1.07 at 373°K
(Lengyel and Conway, 1983
), because the extent of hydrogen bonding in water is decreased by thermal motion (Ewell and Eyring, 1937
; Morgan and Warren, 1938
). The validity of the common practice of subtracting hydrodynamic from total to obtain the "excess" H+
conductance has been questioned recently, on the basis that little conventional hydrodynamic conductance
can occur due to the strength of first-shell hydrogen
bonds, which trap the H3O+ ion in a densely hydrogen-bonded network of water molecules (Agmon, 1996
). In
this view, all H+ conduction occurs by prototropic
transfer. H+ permeation through channels most likely
occurs by this mechanism.
Permeation is not equivalent to diffusion in bulk solution.
The conductance of most cation channels is weakly
temperature sensitive (Table II). A higher Ea has been
reported for a lymphocyte K+ channel, 8.2 kcal/mol or
twice that of free diffusion (Lee and Deutsch, 1990),
and even higher values for inward rectifier K+ channels, although these higher values are from macroscopic measurements that may reflect factors other
than changes in single-channel current amplitude. The
Q10 of the conductivity of physiological monovalent cations (Na+ and K+) in aqueous solutions is ~1.2-1.3 between 5 and 35°C, while that of H+ is only 1.14-1.20
(Robinson and Stokes, 1959
). The similarity of the Q10
for aqueous diffusion of ions and open channel conductance suggests that ions permeate channels in an
environment approximating aqueous diffusion (e.g.,
Horn, 1984
; Stein, 1986
). This implies that there are
not large energy barriers in the permeation pathway, and the ion should permeate at a rate roughly comparable with its diffusion in bulk solution. The conductance of various channels is somewhat less than calculated from the bulk diffusion coefficient, given the dimensions of the pore (Stein, 1986
). Calculated using the Poisson-Nernst-Planck approach, the effective diffusion coefficient of ions inside channels during permeation is roughly an order of magnitude lower than in
bulk solution (Chen et al., 1997
). Part of the higher Ea
for conduction through ion channels than bulk solution may reflect the energetic cost of partial dehydration of the ion; the effective hydration number of ions
in solution decreases at higher temperatures, facilitating entry into the channel (Kuyucak and Chung,
1994
). The mobility of H+ in gramicidin channels,
which are single-file water-filled pores (Levitt et al.,
1978
; Finkelstein and Andersen, 1981
), at high [HCl] is
not much lower than in bulk HCl solution, suggesting that this water-filled pore offers little additional intrinsic resistance (Cukierman et al., 1997
). H+ current
through single gramicidin channels has a Q10 of 1.33 (4.8 kcal/mol) at 1 M HCl (Akeson and Deamer,
1991
), about twice that of the mobility of H+ in bulk solution. Akeson and Deamer (1991)
speculated that misaligned hydrogen bonds between waters in the pore
might increase the Ea for proton hopping. The recent
proposal that the rate-determining step in H+ conduction in water is the breaking of second hydration shell hydrogen bonds of strength 2.5 kcal/mol (Agmon,
1995
) leads to the idea that entry of H+ into a channel
must require breaking a first shell hydrogen bond, which would be about twice as strong (Agmon, 1996
),
consistent with the conclusion that H+ permeation
through gramicidin is entirely diffusion limited (Decker and Levitt, 1988
). The Q10 for H+ permeation reported
here is substantially larger, especially considering the
uniquely low Q10 of H+ conduction in aqueous solution. Evidently, the rate-limiting step in H+ permeation
through voltage-gated H+ channels is thermodynamically distinct from diffusion.
What is the rate-limiting step in permeation?
Table III compares Ea for voltage-gated H+ channels with various
processes involving protons. We focus on parameters
related to hydrogen bonding and to the two separate
processes believed to be required for H+ passage
through an HBC, namely a hopping step (ionic defect
migration) and a turning step (Bjerrum L defect migration, or reorientation/rotation of the elements in the
HBC to "reload" for the next H+) (Nagle and Morowitz, 1978; Nagle and Tristram-Nagle, 1983
). The Ea
found for H+ permeation through voltage-gated channels, 18-27 kcal/mol, is much larger than the Ea for H+
diffusion, 2.6 kcal/mol, and more than double the free
energy of water rotation in ice, 8.4 kcal/mol (Glasstone
et al., 1941
). Of the few processes in Table III with Ea in
the range observed here, hydrolysis is intriguing because Kasianowicz et al. (1987)
proposed this mechanism to account for the problematic supply of sufficient
protons to support the large H+ fluxes observed in protonophore-doped membranes. Voltage-gated H+ currents verge on being large enough to require this or
some other special mechanism to supply H+ to the
channel (DeCoursey and Cherny, 1996b
).
Could hydrolysis provide enough protons to conduct H+ current?
The hydrolysis mechanism is effectively a proton
transfer reaction in the energetically unfavorable direction from water, with pKa 15.7, to a hypothetical protonation site at the inner mouth of the channel, whose pKa
is unknown but likely much lower. If we assume that
the reverse reaction is rapid and diffusion limited and
occurs at 2.3 × 1010 M1 s
1 (Eigen and Hammes,
1963
), then the forward reaction rate is determined by
the pKa of the channel (Bell, 1973
; Eigen, 1964
). The
maximum single-channel H+ current assuming that all
protons come from hydrolysis is then only 0.04 fA for
His (histidine); pKa 6.0), 6.2 fA for Cys (cysteine; pKa
8.18), and 477 fA for Tyr (tyrosine; pKa 10.07). The elementary current through H+ channels in neutrophils
was estimated at ~1 fA (DeCoursey and Cherny, 1993
),
and more recent estimates are somewhat larger (V.V. Cherny and T.E. DeCoursey, unpublished observations). Thus the hypothesis that hydrolysis provides
most of the protons is plausible only if the entry site has
an effective pKa
8 (i.e., Cys, Tyr, Lys, Arg). A speculative mechanism that might overcome the limited intrinsic rate of hydrolysis assumes that the H+ channel is a
metalloprotein. The zinc ion at the catalytic center of
carbonic anhydrase greatly lowers the pKa of the H2O
molecule bound to it, facilitating hydrolysis, as well as
electrostatically repelling the nascent proton from the
resulting OH
(Liang and Lipscomb, 1988
). An attractive feature of hydrolysis is that it would occur at a rate
relatively independent of pH, and this could account
for the near pH independence of the gH (Byerly et al.,
1984
; Demaurex et al., 1993
; DeCoursey and Cherny, 1994b
, 1995
, 1996a
; Cherny et al., 1995
).
Entry of the proton into the HBC is not rate limiting.
Entry
of the ionic defect into an HBC should have a substantial Ea (Nagle et al., 1980; Table III), comprising the
Born electrostatic energy required to insert a charge
into a low dielectric membrane/channel and the chemical energy required to protonate a group on the HBC (John F. Nagle, personal communication). The transmembrane movement of each electronic charge e during H+ conduction across an HBC is divided into two
parts, reducing the Born energy. In ice (Scheiner and
Nagle, 1983
), 0.64 e is carried across during the H+
hopping step and the other 0.36 e is carried during the
"turning" step as the HBC reorients to permit the next
transfer. The Born energy f 2q 2/(2 a
) amounts to 7 kcal/mol under reasonable assumptions (Nagle et al.,
1980
): the fractional charge f = 0.5 because we do not know the nature of the HBC, q = the electronic charge,
ionic radius a = 3 Å, and the dielectric constant
= 2. The chemical energy for a proton to enter the HBC is
(pH
pK) × 1.34 kcal/mol. If the group that comprises the mouth of the channel has a much lower pKa
than the ambient pH, entry will be thermodynamically
unfavorable and require a high Ea. Generating the
large Ea that we observe (Tables IB and III by this
mechanism would require a large difference (pHi
pK) on the order of 8-15 U. The forward rate constant
for channel protonation would be too low to result in
much current, and we therefore rule out the entry step
as rate limiting in permeation.
|
Permeation is rate determining.
Many types of evidence
point to H+ permeation through the channel as being
rate determining for voltage-gated H+ channel current.
Even if hydrolysis could provide sufficient protons, it
seems unlikely to be rate limiting. IH does not saturate at large voltages except at low intracellular buffer concentration, and in this situation the apparent saturation probably reflects bulk pHi changes (DeCoursey,
1991). If increasing the voltage increases the current,
then the rate-determining step must be voltage dependent. Entry into or exit from the pore could conceivably appear voltage dependent if permeation occurred
as a collective event, a possibility suggested by the high
proton polarizability of numerous hydrogen-bonded
systems (Zundel, 1992
). However, entry/exit could not
appear voltage dependent if this were the rate-limiting step. Permeation across an HBC might involve a large
Ea. Because both the hopping and turning steps in
HBC conduction carry fractional charges (Scheiner
and Nagle, 1983
), both would be voltage dependent and either could be rate determining.
Are H+ channels water-filled pores?
We have suggested
that voltage-gated H+ channels are unlikely to be water-filled membrane-spanning pores (DeCoursey and Cherny, 1994b, 1995
, 1997
; Cherny et al., 1995
). Three types of
evidence support this conclusion. (a) Voltage-gated H+
channels are extremely selective, with no detectable
permeability to cations other than H+ or D+, and permeability ratios PH/Pcation > 106
108, calculated from
deviations of Vrev from EH (Kapus et al., 1993
; Demaurex et al., 1993
; DeCoursey and Cherny, 1994b
, 1997
;
Cherny et al., 1995
). A water-filled pore would be expected to have detectable permeability to other cations
because H3O+ and K+ have nearly identical radii. (b)
The unitary H+ channel conductance appears to be
near saturation at pH 7.5, eight orders of magnitude
lower [H+] than at saturation of H+ current through
other water-filled ion channels (DeCoursey and Cherny,
1994b
). (c) The H+/D+ conductance ratio is 1.9 at 20°C
(DeCoursey and Cherny, 1997
), much greater than
1.35 in the prototypical water-filled channel gramicidin
(Akeson and Deamer, 1991
). Some uncertainty is introduced by the possibility that the water molecules in a
pore are constrained by interactions with the walls
(e.g., Nagle et al., 1980
; Gutman et al., 1992
). Molecular dynamics simulations indicate that rotational relaxation rates for water molecules confined inside narrow
channels are reduced compared with bulk (Sansom et
al., 1996
). Ab initio molecular orbital calculations
(Scheiner, 1981
) show that the Ea for proton transfer
between hydrogen-bonded chains of water molecules
increases dramatically and superlinearly if the inter- oxygen distance is increased or the bond angles are deformed, and theoretically could be as large as found
here. The deuterium isotope effect on conduction is
much larger in ice than in liquid water (Kunst and Warman, 1980
). Table III shows that the Ea for some of the
steps likely involved in H+ conduction through a water-filled pore (water rotation, proton hopping, and defect
migration) is increased severalfold in ice. Even so, the
Ea observed here for IH remains about double any of
these values, suggesting that if the H+ channel were a
water-filled pore, then it must constrain the water molecules more tightly than in ice. a and b above remain hard to reconcile with the idea of a water-filled pore, although waters rigidly frozen inside the channel could
prevent other cations from permeating. On balance,
we favor the idea that the channel does not contain a
continuous chain of waters that span the membrane.
Hydrogen-bonded chain.
The alternative to a water-filled
pore is a hydrogen-bonded-chain, a continuous "proton wire" comprising some combination of side groups
of amino acids and possibly intercalated water molecules. (We imply the presence of substituents other
than water in our use of the term HBC, although a
pure "water wire" is formally just a special type of
HBC). The HBC mechanism was proposed by Nagle
and colleagues for other biological H+ channels such
as those in bacteriorhodopsin and in H+ATPases of mitochondria and chloroplasts (Nagle and Morowitz, 1978; Nagle and Tristram-Nagle, 1983
). Comparison of
the Q10 for H+ translocation through other membrane
proteins (Table IV) generally suggests that H+ transporters that involve conduction via hydrogen-bonded
chain mechanisms that include protein groups (such as
bacteriorhodopsin, the H+ channel of proton pumps,
and MotA) tend to have substantially higher Q10 than
does H+ permeation through the water-filled gramicidin pore. The high Q10 observed here for IH suggests
the existence of significant energy barriers in the permeation pathway. Although we consider a simple water
wire to be unlikely, the hydrogen-bonded chain comprising the permeation pathway certainly could include
intercalated water molecules, as demonstrated recently
for bacteriorhodopsin (Pebay-Peyroula et al., 1997
).
The M2 viral proton channel may comprise a water-filled pore with a single constriction, occluded by His
groups that shuttle protons by a tautomerization (ring flipping) mechanism (Pinto et al., 1997
). Although the
temperature dependence of its conductance is not
known, the rate-determining step in CO2 catalysis by
carbonic anhydrase is an intramolecular proton transfer from Zn-bound water to His (Liang and Lipscomb, 1988
; Taoka et al., 1994
), with Ea ~8 kcal/mol (Ghannam et al., 1986
). In summary, the high Q10 of H+ permeation strengthens the case that the voltage-gated H+
channel is not a water-filled pore like other ion channels. Entry into the channel may consist of simple protonation of the end of an HBC. This may explain the
paucity of inhibitors of this conductance
there is no
pore to occlude. The main inhibitors of H+ currents
are polyvalent cations, which may bind to the putative proton entry site or near enough to it to lower the local
[H+] electrostatically.
|
Physiological relevance.
At room temperature, mammalian voltage-gated H+ channels open very slowly in
comparison with most ion channels, although other slow channels exist, such as the cardiac delayed rectifier. Mammalian H+ channels and those in molluscan
neurons differ mainly in activation kinetics (DeCoursey, 1991, 1998
), the latter opening in a few milliseconds (Byerly et al., 1984
). The strong temperature dependence of gating means that mammalian H+ channels activate 20-40× faster at body temperature than at
room temperature. When the high Q10 of IH is also considered, it is clear that H+ channels are capable of
rapid and highly efficient acid extrusion. The combined effect of the high Q10 for IH and for gating means that the gH will be activated much faster at body temperature than at room temperature, where most studies
of H+ currents have been done.
Comparisons among different cells and mammalian species.
A final point to emerge from this study is that the temperature sensitivity of H+ currents and several kinetic
measures of H+ channel gating are quite similar in human neutrophils, THP-1 monocytes, and promyelocytic
HL-60 cells, mouse BV-2 microglial cells, and rat alveolar epithelial cells and macrophages. A recent study reported highly temperature sensitive H+ currents in murine mast cells, with a Q10 of 6.0 or 9.9 for the outward
current at the end of voltage ramps at pHi 5.5 or 7.3, respectively (Kuno et al., 1997). This high sensitivity appeared to distinguish the H+ channel in mast cells from
that in other cells for which little data had been published. However, the H+ current amplitude at the end
of a voltage ramp depends on both the conductance
and gating kinetics
at low temperatures, fewer channels will open during the ramp. In the present study,
we distinguished between effects of temperature on
gating and on open channel conductance. Once this
separation was made, it became apparent that the temperature sensitivity of both conductance and gating is
similar in all cells in which these properties have been
studied. Although there are distinct varieties of H+
channels (DeCoursey, 1998
), their temperature sensitivity does not differ obviously. Byerly and Suen (1989)
reported a Q10 of 2.1 for whole-cell IH in snail neurons.
Because activation is very rapid in Lymnaea, this value is
a relatively pure reflection of IH uncontaminated by
gating effects, and is well within the range observed for
the Q10 of IH in mammalian cells (Table IA). These observations provide no evidence for significant differences in either the mechanism of H+ permeation or
the rate-determining steps in gating in various types of
H+ channels.
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FOOTNOTES |
---|
Address correspondence to Tom 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
Original version received 1 June 1998 and accepted version received 20 July 1998.
Portions of this work were previously published in abstract form (Cherny, V.V., and T.E. DeCoursey. 1998. Biophys. J. 74:A316).The authors thank Fred S. Cohen, John J. Kasianowicz, Vladislav S. Markin, and Fred N. Quandt for thoughtful comments on the manuscript. The authors are especially grateful to John F. Nagle for explaining the assumptions used to estimate the free energy cost of defect entry into an HBC, and to Richard W. Aldrich for suggestions about gating schemes.
This work was supported most recently by research grant HL-52671 to Dr. DeCoursey from the National Institutes of Health (NIH). Earlier studies were supported by a Grant-in-Aid to Dr. DeCoursey from the American Heart Association with funds contributed by the American Heart Association of Metropolitan Chicago, and before that, by research grant HL-37500 and Research Career Development Award K041928 to Dr. DeCoursey from the NIH.
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Abbreviations used in this paper: |
---|
E a, activation energy; EH, Nernst potential for H+; gH, H+ chord conductance; HBC, hydrogen-bonded chain; IH, extrapolated H+ current amplitude; Vhold, holding potential; Vrev, measured reversal potential.
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