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J. Biol. Chem., Vol. 275, Issue 40, 31038-31050, October 6, 2000
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From the
Received for publication, May 1, 2000, and in revised form, July 24, 2000
The M2 ion channel protein of
influenza A virus is essential for mediating protein-protein
dissociation during the virus uncoating process that occurs when the
virus is in the acidic environment of the lumen of the secondary
endosome. The difficulty of determining the ion selectivity of this
minimalistic ion channel is due in part to the fact that the channel
activity is so great that it causes local acidification in the
expressing cells and a consequent alteration of reversal voltage,
Vrev. We have confirmed the high proton selectivity of the
channel (1.5-2.0 × 106) in both oocytes and
mammalian cells by using four methods as follows: 1) comparison of
Vrev with proton equilibrium potential; 2) measurement of
pHin and Vrev while
Na+out was replaced; 3) measurements with
limiting external buffer concentration to limit proton currents
specifically; and 4) comparison of measurements of
M2-expressing cells with cells exposed to a protonophore.
Increased currents at low pHout are due to true activation
and not merely increased [H+]out because
increased pHout stops the outward current of acidified cells. Although the proton conductance is the biologically relevant conductance in an influenza virus-infected cell, experiments employing methods 1-3 show that the channel is also capable of conducting NH4+, probably
by a different mechanism from H+.
The M2 protein of influenza A virus is thought to
function as an ion channel that permits protons to enter virus
particles during virion uncoating in endosomes. In addition, in
influenza virus-infected cells, the M2 protein causes the
equilibration of pH between the acidic lumen of the trans-Golgi network
and the cytoplasm (reviewed in Refs. 1 and 2). The activity of the
M2 ion channel is inhibited by the antiviral drug
amantadine (3-5). The mature M2 protein consists of a
23-residue N-terminal extracellular domain, a single internal
hydrophobic domain of 19 residues that acts as a transmembrane domain
and forms the pore of the channel, and a 54-residue cytoplasmic tail
(6). Chemical cross-linking studies showed the M2 protein
to exist minimally as a homotetramer (7-9). Statistical analysis of
the ion channel activity of mixed oligomers also indicated that a homotetramer is the minimal active oligomeric form of the protein (10).
Despite the small size of the active M2 oligomer, several
lines of evidence indicate that ion channel activity is intrinsic to
the M2 protein. First, ion channel activity has been
observed in three different expression systems, Xenopus
oocytes (3, 11, 12), mammalian cells (5, 13), and yeast (14). Second, M2 channel activity has also been recorded in artificial
lipid bilayers from a reconstituted peptide corresponding to the
transmembrane domain of the M2 protein (15) and from
purified M2 protein (16). Thus, due to its structural
simplicity, the M2 ion channel is a potentially useful
model for the study of ion channels in general.
Although a great deal of evidence indicates H+ is the
biologically relevant ion for the role of M2 protein in the
life cycle of the influenza virus (1, 3, 17-22), other ions have been shown to be capable of flowing through the channel (12). In addition,
the ion selectivity measured for the M2 channel has been
found to differ depending on whether the activity was measured in
Xenopus oocytes or mammalian cells. When M2
protein was expressed in oocytes, Vrev was found to differ
from the proton equilibrium potential, EH+ as
[H+]out was varied (12). On the other hand,
when M2 protein was expressed in MEL cells,
Vrev was found to agree with EH+ (5). In a recent study (23), we found IH+ of the
M2 ion channel to be so large that it was capable of
decreasing [H+]out in the locale of the
extracellular pore of the channel if the expressing cells were bathed
in medium of low buffer concentration. One possible explanation for the
different results may be that the channel is also capable of acidifying
the interior of some expressing cells, thereby altering reversal
voltage, Vrev. Shimbo and co-workers (12) found that
replacement of Na+ with Li+ decreased currents,
and replacement of Na+ with
NH4+ increased currents. In principle,
these effects could have resulted from one of two mechanisms. Either
these ions affected the proton current, IH+, or the
replacing ions permeated the M2 ion channel. In this study,
we were able to study the effects of these ion replacements on proton
currents specifically by taking advantage of the finding that inward
H+ currents are limited when the concentration of buffer in
the bathing medium is reduced (23) to distinguish between these possibilities.
The M2 ion channel current is increased in amplitude when
the pH of the extracellular domain is lowered (3, 5, 24). This increase
in current occurs within the range of pH values expected for titration
of histidine (24). The only amino acid in the transmembrane domain of
the M2 protein with a titratable group in this pH range is
His37, and when His37 is replaced by
Ala, Gly, or Glu, the proton selectivity of the channel is greatly
reduced, and the channel is conductive over a wider range of pH (3,
24). It has been proposed that His37 forms a
selectivity filter for protons and that H+ conduction may
occur by tautomerization of the imidazole side chain of
His37 (25). Although the H+ current of
the M2 ion channel protein is increased by elevated [H+] in the extracellular medium, this increased current
may be due to either the increased abundance of the conducting species
or activation of the channel at low pH, or both factors operating together. One way to distinguish pH-dependent changes in
activity from the effects of increased abundance of H+ at
low pH is to compare the efflux of H+ from acidified cells
that express the M2 protein to the efflux from acidified
cells treated with the electrogenic protonophore FCCP.1 Cell acidification can
be achieved by lowering the pH of the medium bathing
M2-expressing or FCCP-treated cells. If the M2 ion channel is indeed activated by low pHout and conversely
deactivated by neutral or alkaline pHout, then the efflux
of H+ should be greater for FCCP-treated cells than for
M2-expressing cells upon return to a bathing solution of
neutral or alkaline pH.
In this study we measured ionic currents and pHin in two
M2 expression systems to ensure that the results obtained
were not specific to the cell type. The results demonstrate that under normal physiological conditions the M2 ion channel
specifically conducts H+. We also demonstrate that
NH4+ can permeate the channel, by a
mechanism that differs from that for H+ permeation.
Furthermore, by comparison of the outward currents of acidified,
M2-expressing cells and FCCP-treated cells, we confirm that
M2 ion channel activity is modulated by the pH of the
solution bathing the extracellular N-terminal domain of the channel.
mRNA Synthesis--
The cDNA to the A/Udorn/72 mRNA
was cloned into the BamHI site of pGEM3 such that mRNA
sense transcripts could be generated by using the bacteriophage T7 RNA
polymerase promoter and T7 RNA polymerase. For in vitro
transcription, plasmid DNAs were linearized downstream of the T7
promoter and the M2 cDNA with XbaI. In
vitro synthesis and quantification of
7mG(5')ppp(5')G-capped mRNA was carried out as
described previously (3).
Culture and Microinjection of Oocytes--
Oocytes were removed
from female Xenopus laevis (Nasco, Fort Atkinson, WI),
defolliculated by treatment with collagenase B (2 mg/ml; Roche
Molecular Biochemicals), and incubated in ND96 (96 mM NaCl,
2 mM KCl, 3.6 mM CaCl2, 1 mM MgCl2, 2.5 mM pyruvic acid, 5 mg/ml gentamicin, 5 mM HEPES, pH 7.5, osmolality ~210 mosmol/kg) at 19 °C. Oocytes at stage V were microinjected
with 50 nl of mRNA (1 ng/nl) on the day after defolliculation,
incubated for 24 h in ND96, pH 7.5, and finally incubated for
24 h in ND96, pH 8.5, at 19 °C before use.
Culture and Infection of CV-1 Cells--
CV-1 cells were
cultured and infected with recombinant simian virus 40 expressing the
M2 protein from influenza A/Udorn/72 (rSV40-M2), as described previously (13). Briefly, CV-1
cells grown to confluency at 37 °C, 5% CO2 in culture
media (Dulbecco's modified Eagle's medium + 10% fetal calf serum + penicillin + streptomycin) were trypsinized, pelleted, and resuspended
in culture medium. Resuspended cells were incubated in the presence of
high titer SV40-M2 (100 µl of resuspended CV-1 cells + 1 ml of virus stock) for 4 h. Infected cells were then diluted 1:1
in culture medium and seeded onto 5-mm square glass coverslips arranged
in 3.5-cm Petri dishes (2 ml total volume/dish). Infected cells were then incubated for 48 h before recording to ensure adequate
M2 protein expression.
Measurement of Membrane Current of CV-1
Cells--
M2 currents were recorded from CV-1 cells using
the whole cell patch clamp technique as described previously (13).
Briefly, patch pipettes having tip diameters of ~2-3 µm were
pulled from borosilicate capillary glass, fire-polished, and then
partially filled with pipette solution which contained, in
mM, 145 KCl, 5 EGTA, 1 MgCl2, 5 NaCl, 15 HEPES,
pH 7.4, adjusted with KOH, osmolality 300-310 mosmol/Kg.
Pipettes filled with this solution typically had resistances of ~3-4
M Measurement of Membrane Current of Oocytes--
Whole cell
currents were measured using a two-electrode voltage clamp. Electrodes
were filled with 3 M KCl, and the oocytes were bathed in
either Barth's solution, which contained, in mM, 88 NaCl,
1 KCl, 2.4 NaHCO3, 0.3 NaNO3, 0.71 CaCl2, 0.82 MgSO4, 15 HEPES, pH 7.5, osmolality
~210 mosmol/kg or a modified solution during the recording.
Continuous current-voltage (I-V) relationships were measured with ramps
of membrane voltage since the M2 channel shows no rapid
voltage- or time-dependent gating. These ramps typically
spanned a range of 120 mV in 2 s. Oocyte holding potential was
Measurement of pHin of Oocytes--
Microelectrodes
were silanized and filled with protonophore as described previously
(12). The electrodes were calibrated before each experiment with four
pH values spanning the range encountered during the experiment. The
response time of these electrodes, determined with a stepping motor
device that changed solution pH within 100 ms, was less than 10 ms.
Measurement of pHin of CV-1 Cells--
We used the
fluorometric indicator
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester
(BCECF-AM) to measure pHin. Control cells or cells
infected with rSV40-M2 were incubated (37 °C for 1 h) in a solution containing BCECF-AM in 0.25% Me2SO carrier with a final dye concentration of 0.25 µg/ml. These cells were placed on the stage of an epifluorescence microscope equipped with
a × 20, 0.75 NA (Nikon) objective that allowed up to seven CV-1
cells to be imaged in its field at one time, an intensified CCD camera
and MagiCal image analysis software (Applied Imaging, Sunderland, UK).
The dye was excited with 490 nm illumination to observe pH-induced
changes in fluorescence. To measure the intracellular concentration of
dye and thus allow calibration of pHin of the CV-1 cells,
illumination was applied at 435 nm (the isosbestic wavelength) at the
beginning and end of the measurements from one field of cells. Emission
was recorded at 520 nm. Calibration of the pHin from
fluorescence measurements was done using the FCCP equilibration method
(26). Briefly, the cells were treated with the protonophore FCCP to
allow equilibration of the [H+] across the plasma
membrane. The emission at 520 nm as a result of excitation at 490 and
435 nm was measured while the cell was bathed in solutions with pH
spanning the range of pH values expected to be encountered during the
measurements (pH 4.0, pH 6.7, and pH 9.0), and the resulting ratios
(F490/F435) were used to
construct a calibration curve (see Equation 1 of Ref. 26).
Reversal Voltage Changes within a Few Seconds after Lowering the pH
of the Bathing Solution
The M2 protein from influenza A/Udorn/72 virus was
used for this study. If the M2 ion channel is highly
proton-selective, then the reversal voltage of the current
(Vrev) should change according to the equilibrium potential
for H+ (EH+) when the difference
between pHin and pHout is altered. The reversal
voltage of the currents of cells expressing the M2 protein
can be measured from continuous current-voltage relationships measured
with ramps of membrane voltage because the M2 ion channel is not voltage-activated on the time scale of the ramps of voltage that
are practical to use.
Oocytes--
We measured the Vrev of
amantadine-sensitive currents in M2-expressing
Xenopus oocytes at 20-s intervals using two-microelectrode voltage clamp. Oocytes whose membrane voltage was clamped to CV-1 Cells--
The membrane currents and Vrev were
measured using the whole cell patch clamp technique in CV-1 cells that
expressed the M2 protein (Fig.
2), whereas pHout was reduced
in less than 100 ms from pH 7.4 to pH 6.2 with a stepping-motor device
(see "Experimental Procedures"). The pHin of CV-1
cells, measured fluorimetrically in a separate experiment described
below, was used to calculate EH+. The peak
Vrev measured in a solution of pH 6.2 was 41.8 ± 3.5 mV S.E. (n = 12), and EH+ was
38.9 ± 4.7 mV S.E. (n = 13). In less than 1 min
after lowering pH of the bathing solution, Vrev reached a
peak near EH+ and then returned to more negative values (Fig. 2). We calculated the permeability of H+
relative to that for Na+ assuming 2 mM residual
intracellular Na+ concentration for these CV-1 cells, using
the GHK equation, and found that the relative permeability was about
1.8 × 106.
Since Vrev changed after reaching a peak in the low pH
solutions in a rapid and systematic manner in both
M2-expressing CV-1 cells and oocytes, we studied the reason
for the systematic changes in Vrev, and we employed other
means to study the ion selectivity of the channel. There are a number
of possible explanations for the decrease of inward current and the
return of Vrev to more negative voltages after reaching a
peak value that was observed in M2-expressing cells bathed
in low pH solutions. 1) The influx of ions through the M2
channel could activate an endogenous outward current that opposes the
current flowing through the M2 channel. 2) The
M2 ion channel might undergo an activity dependent change in ion selectivity. 3) The M2 ion channel might inactivate
after long periods in low pH solutions. 4) A constant influx of protons through the M2 channel might cause acidification of the
cell cytoplasm and thus decrease the driving force on protons.
For explanations 1 and 2, the possibility was tested that the shift in
Vrev observed in oocytes and CV-1 cells might be due to
activation of an amantadine-insensitive, endogenous current or an
activity-dependent change in ion selectivity by measuring currents under ionic conditions chosen to minimize all but
H+ currents. The experiments were conducted in CV-1 cells,
for which it was possible to control the composition of both the
intracellular and extracellular solutions. We found that it was not
possible to perform these experiments by changing the internal
composition in oocytes using the cut-open technique because small leaks
that developed were indistinguishable from M2 currents,
except by application of amantadine, the effects of which are not
reversible on the time scale of these experiments. The principal
endogenous currents of CV-1 cells were found to be similar to those of
HEK293 cells, i.e. inward Cl
For explanation 3, it was also unlikely that the decrease of current
and negative shift of Vrev observed in low pH was the result of inactivation of the M2 ion channel. This is
because the slope conductance of the I-V relationship actually
increased in both oocytes and CV-1 cells during the shift
(Figs. 1B and 2). This observation is the opposite of what
would have been expected if the channel had been inactivating. The
remaining and most likely explanation for the return of
Vrev to more negative values for cells bathed in low pH
solutions was that intracellular acidification occurred.
For explanation 4, we tested whether the return of Vrev to
negative potentials and the decrease in amplitude of the inward current
observed after their peak values occurred were the result of cell
acidification by measuring the shift under conditions designed to
minimize acidification. These conditions were achieved as discussed in
the following two paragraphs.
If the return of Vrev to more negative values is due to
acidification, then a larger negative shift should be observed when there is a larger inward driving force on H+. This can be
obtained either by lowering the pH of the bathing solution further or
by making the holding voltage still more negative than
EH+. Since most cells become unstable in very
low pH solutions (<pH 5.5), we decided to modulate the size of the
inward current of oocytes by varying the cell holding voltage while the cells were bathed in Barth's solution of pH 5.8 (Fig.
3). Holding oocytes at voltages more
negative than
To minimize the acidification of M2-expressing cells in low
pH solutions, the intracellular buffer concentration was increased. Again, as the ionic composition of the ooplasm could not be controlled, this experiment was performed in M2-expressing CV-1 cells
using the whole cell patch clamp technique. We studied the effect of elevated concentrations of the buffer of the pipette solution. The
results obtained from pipettes containing 15 and 120 mM
HEPES buffer on Vrev and the amplitude of the inward
current as pH was lowered from pH 7.4 to pH 6.2 were compared. It was
observed that for both 15 and 120 mM buffer in the pipette,
lowering the external pH led to an increase of inward current in cells
held at Measurements of pHin during Exposure to Low
pHout
It was found with direct measurements of pHin that the
return of Vrev to negative values observed in
M2-expressing cells bathed in solutions of low pH was
indeed accompanied by cell acidification.
For Oocytes--
Intracellular pH was measured using an electrode,
and the cells were voltage-clamped to measure the membrane current,
Vrev, and membrane conductance (Fig.
4A). Lowering the
pHout from pH 8.5 to pH 5.8 produced a large inward current
and a rapid shift of Vrev to more positive values, as noted
earlier (Fig. 1). Immediately after reaching a peak, the amplitude of
the inward current began to decrease. The Vrev reached a
peak value prior to the amplitude of the inward current and returned to
more negative voltages as the amplitude of the inward current
decreased. Measurement of pHin during this time (Fig.
4A) revealed that pHin did not change immediately after introduction of low pH bathing solution, despite the
presence of a detectable inward current. However, after ~120 s
bathing in low pH solution, just after the amplitude of the inward
current reached a maximum, pHin began to decrease steadily. The onset of this acidification lagged the decrease of inward current
and negative shift of Vrev by ~100 s. Plots of
Vrev versus pHin (Fig.
4B) revealed that the initial shift of Vrev to
more positive values and subsequent return to more negative values occurred independently of changes in pHin that were
recorded with a pH microelectrode. After ~150 s in the low pH
solution, the observed changes in Vrev and pHin
occurred together. Recovery of pHin to control values was
observed both when the pH of the bathing solution was returned from pH
5.8 to pH 8.5 and also when 100 µM amantadine was added
to the solution (pH 5.8). The recovery of pH under both of these
conditions followed an attenuation of the inward current. These results
demonstrate that oocytes expressing the M2 protein acidify
when there is a large inward H+ current. This
H+ influx causes an acidification of the cytoplasmic
solution accessible to the pH microelectrode after a delay with respect
to the time when Vrev reaches its peak value. These results
are consistent with a diffusional delay for H+ in the
cytoplasm of the expressing cells, between the membrane and the
location of the tip of the pH electrode. This would result in a delay
between a decrease of pH at the cytoplasmic opening of the pore of the
M2 channel, which determines Vrev, and the decrease of pH at the tip of the pH microelectrode.
For CV-1 Cells--
pHin was measured by ratiometric
imaging of the fluorescence of the pH-sensitive indicator BCECF. Cells
were infected with rSV40-M2 and loaded with BCECF-AM prior
to measuring fluorescence. The pHin of
M2-expressing cells measured in medium of pH 7.4 was pH
6.87 ± 0.81 S.E. (n = 8) in one experiment and pH
7.04 ± 0.09 S.E. (n = 5) in a second experiment.
When the pH of the bathing medium was lowered from pH 7.4 to pH 6.2 for
~200 s, the M2-expressing cells underwent a rapid
decrease in pHin by 0.63 pH units (± 0.056 pH units S.E.,
n = 8) in one experiment and by 1.07 pH units (± 0.085 pH units S.E., n = 4) in a second experiment. These
changes were reversible upon return to bathing medium of pH 7.4 (Fig. 5A). Cells treated with
amantadine did not undergo this change in pHin when bathed
in medium of pH 6.2. The membrane voltage of the CV-1 cells expressing
the M2 protein was not clamped in these measurements, and
thus it was possible that alterations of driving force might have
influenced the membrane proton currents. To control membrane voltage,
we took advantage of the potassium ionophore valinomycin to help
maintain the membrane voltage at a value determined by the ratio of
[K+] across the membrane. This was done by introducing
valinomycin (20 µM final concentration in 0.02%
Me2SO carrier; exposure to carrier alone produced no
changes in fluorescence) into the bathing medium. It was found that the
rate of acidification increased for lower [K+] of the
bathing medium, consistent with increased driving force for protons
caused by a more negative membrane potential (Fig. 5B).
The systematic variation of Vrev observed with both
M2-expressing oocytes and CV-1 cells has important
implications for determining the ion selectivity of this channel. When
measured in low pH solutions, the value obtained for Vrev
will depend upon the time when it is measured. This time dependence of
Vrev is probably the result of acidification of the bulk
solution accessible to the cytoplasmic mouth of the pore of the
M2 ion channel. Thus, even the most appropriate measurement, that of the peak value of Vrev, is likely to
be distorted by acidification.
Ion Substitution Studies
The possibility that the inward current of the M2 ion
channel might in part be carried by ions other than the proton was
tested by replacing other extracellular ions with large, presumably
impermeant, ions. We tested for Na+ permeability by
replacing Na+, the major extracellular cation, with other
ions. These experiments were performed in oocytes. We also replaced
NaCl with mannitol. Changes in the peak Vrev, conductance,
and pH were measured after reducing pH of the bathing medium. The
principle of this experiment is that if Na+ normally flows
through the channel, replacing Na+ with an impermeant
cation should decrease the amplitude of the inward current but not
change the acidification rate. NaCl in the extracellular medium was
replaced with equimolar concentrations of
N-methyl-D-glucamine Cl, LiCl,
NH4Cl, or iso-osmotically with mannitol. To determine the
effect of these Na+ substitutions, we first measured
pHin, conductance, and Vrev in the control
solution (containing NaCl), at both pH 8.5 and pH 5.8. This was
followed by measurements in a pH 5.8 solution in which NaCl was
replaced. Finally, the measurements were repeated in the control
solution at pH 5.8 to check for reversibility before applying 100 µM amantadine. Oocytes were bathed in control solution of
pH 8.5 between exposures to low pH solutions to allow recovery from
intracellular acidification. A full recovery of pHin to
control values typically took 15-20 min in the pH 8.5 solution. As the M2 ion channel is closed at this pH and there was no
residual current, it is thought that the restoration of
pHin to control values was the result of a non-electrogenic
endogenous H+ exchanger.
It was found that substitution of Na+ with large,
presumably impermeant, cations such as NMDG+ or
replacing NaCl iso-osmotically with mannitol had no detectable effect
on peak Vrev, conductance, or oocyte acidification rate in
low pH solutions (Table I). This result
demonstrates that the M2 ion channel does not
conduct detectable amounts of Na+ ions. However, when NaCl
was replaced with LiCl or NH4Cl the results differed
oppositely from those in control solutions. Replacement of NaCl with
LiCl decreased conductance and acidification rate (Table I) but had no
detectable effect on the peak Vrev (Fig. 6A), and the peak of
Vrev occurred at about the same time as it did in
Na+-containing solutions, about 20 s after changing
solutions. Replacement of NaCl with NH4Cl, on the other
hand, increased conductance, increased acidification rate (Table I),
and shifted Vrev to potentials more positive than those
observed in NaCl (Fig. 6B). The peak of Vrev
also occurred in NH4+-containing
solutions at about the same time as it did in
Na+-containing solutions, about 20 s after changing
solutions. This increase in conductance was fully sensitive to
amantadine (100 µM) and control uninjected oocytes
exposed to NH4+-containing solutions at
pH 6.2 or lower did not display inward currents (in contrast, we have
found that oocytes bathed in NH4+
containing solutions at pH 7.5 or above exhibit large endogenous currents (29)). These results can be interpreted by either
Li+ and NH4+ replacing
Na+ in permeating the pore, flowing independently through
the pore, or Li+ actually interfering with conduction
through the pore. To distinguish among these possibilities, low
external buffer concentrations were used to limit specifically the
component of current carried by protons, IH+.
Currents Measured with Low External Buffer Concentration to Limit
IH+
Advantage was taken of the limitation of H+ currents
that can be achieved for M2-expressing cells by reducing
the buffer concentration of the bathing medium (23). This means was
used to limit H+ currents to determine if the alterations
in amplitude of the M2 current we observed with
Li+ and NH4+ were due to an
effect on H+ currents or due to an effect on other ionic
currents. The limitation of H+ currents from low external
buffer concentration results from a decrease in the [H+]
near the extracellular mouth of the pore of the M2 ion
channel (23). This decrease in [H+] is reflected in a
decrease of current seen during a 2-s-long voltage clamp pulse (Fig.
7). If ion substitution inhibits
H+ conduction through the channel, then the decrement in
amplitude of the inward current during a voltage clamp pulse applied
while bathed in a solution of low buffer concentration should be
proportional to the decrease of current due to the ion replacement. If,
on the other hand, the replacing ion permeates the channel by a
mechanism independent of H+ conduction, then the decrement
in amplitude of the inward current while bathed in a solution of low
buffer concentration should be unaffected by the replacement. The
decrease in amplitude of the inward M2 current was recorded
during a 2-s hyperpolarizing voltage clamp pulse to
Permeation and Activation of the M2 Ion Channel
of Influenza A Virus*
,,
**, and
Department of Neurobiology and Physiology
and the ¶ Department of Biochemistry, Molecular Biology, and
Cell Biology, Howard Hughes Medical Institute, Northwestern
University, Evanston, Illinois 60208-3500 and
§ Institut für Biologische Informationsverarbeitung,
Forschungszentrum, 52425 Juelich, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. CV-1 cells attached to glass coverslips were transferred to a
recording chamber filled with a solution that contained, in
mM, 140 NaCl, 5.3 KCl, 0.55 MgSO4, 1.8 mM CaCl2, 5.5 glucose, 15 HEPES, pH 7.4, or 15 mM MES, pH 6.2, osmolality ~300 mosmol/kg. Seals
(in excess of 10 G) were made by gently pressing the patch pipette
against a CV-1 cell and then applying ~12 mm Hg suction without
delay. The whole cell configuration was achieved by using a brief pulse
of high voltage combined with gentle pipette suction. In the whole cell
configuration, cells had access resistances of <10 M. Cells were
generally bathed in pH 7.4 solution and held at 
20 mV. Whole cell
currents were recorded after the bathing solution was changed from pH
7.4 to pH 6.2 using a Fast Step Perfusion System (model SF77B, Warner Instruments Corp., Hamden, CT). By using this system, solution changes
could be made in less than 100 ms. M2-specific currents were identified by sensitivity to block by 100 µM amantadine.
20 mV unless stated otherwise.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 mV
produced a large amantadine-sensitive inward current (~1 µA) when
the pH of the bathing solution was lowered from pH 8.5 to pH 5.8 (Fig.
1A). During the time when the
oocyte was bathed in low pH medium, Vrev, measured using
voltage ramps, became more positive within a few seconds and reached a
maximum prior to the time when the inward current reached its maximum
amplitude (Fig. 1B). The average peak Vrev,
calculated from the current-voltage relationship of the
amantadine-sensitive current, was 51.4 ± 1.22 mV S.E.
(n = 34), a value more negative than that for
EH+ (85.2 mV ± 1.86 mV S.E.,
n = 34) which was calculated from the known
pHout and the value of pHin measured with a pH
microelectrode at the beginning of the experiment. We calculated the
permeability of H+ relative to that for Na+
assuming 20 mM intracellular Na+ concentration
for these 34 cells, using the Goldman-Hodgkin-Katz (GHK) equation, and
we found that the relative permeability was about 1.5 × 106. However, in two cells that had small
amantadine-sensitive currents, the peak Vrev nearly reached
the value calculated for EH+ (+89 and +91 mV).
At the time when the peak value of Vrev occurred, the
current-voltage relationship was steeper than that obtained at pH 8.5, indicating that the conductance had increased. Shortly after reaching
its peak value, the inward current decreased in amplitude over a period
of a few minutes (Fig. 1A). The current decrease was
accompanied by a return of Vrev back toward more negative
potentials.
View larger version (15K):
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Fig. 1.
Membrane currents and current-voltage
relationships recorded from oocytes expressing the M2
protein. A, time course of the membrane current as the
pH of the bathing medium was lowered from pH 8.5 to pH 5.8 while
membrane voltage was held at 20 mV. Note that the current decreased
spontaneously while the bathing solution was held at pH 5.8 and that
the current was fully inhibited by 100 µM amantadine
(Amant). Upper interrupted line represents zero
current, and lower interrupted line represents maximal
inward current amplitude. Vertical deflections are the result of the
voltage ramps used to measure the current-voltage relationships such as
those shown in B at the times indicated after pH was
reduced. B, current-voltage relationship measured in the
same cell shown in A. Note the return of Vrev to
negative values and the increase in conductance that occurred after 2 min in low pH bathing medium. EH+ is shown with
the vertical interrupted line and was calculated from the
known pHout and pHin measured using an
intracellular pH electrode.
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Fig. 2.
Current-voltage relationships recorded from a
CV-1 cell expressing the M2 protein at pH 7.4 at various
times (numerals above curves) after the pH of the
bathing medium was lowered from pH 7.4 to pH 6.2. Note that the
return of Vrev to more negative values and the increase in
conductance that occurred with time in low pH bathing medium were
faster than those measured in oocytes (Fig. 1B).
EH+ (vertical interrupted line) was
calculated from the known pHout and the pHin
was measured fluorometrically in separate experiments.
and
outward K+ currents (27). Whole cell currents were measured
in CV-1 cells when Cl in the bathing solution was
replaced with methane sulfonate and KCl in the pipette solution with
tetraethylammonium chloride in order to reduce the endogenous currents
of the cells. As before, Vrev was measured at frequent
intervals as the external pH was lowered. Upon lowering the
extracellular pH from pH 7.4 to pH 6.2, a large inward current
developed in M2-expressing CV-1 cells held at 20 mV,
similar to the results obtained with control solutions (Fig. 2). This
inward current was accompanied by a shift of Vrev to
positive values. The peak Vrev (38.8 ± 2.01 mV S.E.,
n = 5) was close to the value of
EH+ (calculated as above, 38.9 ± 4.7 mV
S.E., n = 13). As observed in the control solution, the inward current began to decrease a few seconds after reaching its
maximum amplitude. The decrease of inward current was again accompanied
by a return of Vrev to more negative voltages and an
increase in the slope conductance of the I-V relationship. The fact
that the return of Vrev to negative values still occurred in the absence of other conducting ions suggests that the change of
Vrev was not the result of either activating an endogenous current or an activity-dependent change in ion selectivity.
We calculated the permeability of H+ relative to that for
Na+ using the mean values of 38.8 and 38.9 mV for the
Vrev and EH+, respectively, and
assuming 2 mM residual intracellular Na+
concentration for these CV-1 cells, using the GHK equation, and we
found that the relative permeability was about 2 × 107. However, if the actual values differed by as little as
3 mM, the relative permeability would have been about
2 × 106.
20 mV produced larger inward currents, accelerated the
onset and rate of current decrease, and resulted in a faster rate of
negative shift of Vrev over time (Fig. 3A). It
was also found that Vrev could be directed to more positive
values following the shift in Vrev in low external pH by
making the holding voltage more positive (Fig. 3A).
Furthermore, the return of Vrev to negative values could be
prevented by holding membrane voltage at a large positive value that
was close to EH+ (Fig. 3B). These
results demonstrate that Vrev can be manipulated by holding
voltage, consistent with the current that flows at the holding voltage
being capable of altering pHin of the cell.
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Fig. 3.
Alteration of Vrev as a result of
manipulation of holding potential. A, holding potential
of an oocyte expressing the M2 protein was adjusted to each
of several values (top record) and Vrev was
measured using voltage ramps as shown in Fig. 1B.
B, holding the membrane at +50 mV after reducing
pHout from pH 8.5 to pH 5.8 prevented Vrev from
returning to more negative values. Note that Vrev was
closer to EH+ for holding potentials closer to
EH+ and that Vrev was able to be manipulated
toward both more negative and more positive voltages. Holding at a
membrane voltage of 20 mV caused a rapid decrease in
Vrev. The interrupted line in A and B
shows EH+ calculated from the known
pHout and the pHin measured with an
intracellular pH electrode.
20 mV within a few seconds. It was also observed that for
both 15 and 120 mM buffer in the pipette, after lowering
the external pH, the amplitude of the inward current reached a peak and
then decreased within a few seconds, and during the decrease of inward
current Vrev returned to more negative voltages. Thus,
changes of Vrev could not be prevented by increasing the
intracellular buffer concentration. Reports in the literature show that
in order to control intracellular pH adequately, even with high
concentrations of buffer in the pipette solution, the pipette diameter
must be at least 
of the diameter of the cell (28). The
pipette diameters used in our experiments were on average 3-4 µm
diameter, and the CV-1 cells from which we recorded were ~100-150
µm diameter. The small ratio of pipette diameter to cell diameter
used in these experiments probably explains why even a high buffer
concentration did not stabilize Vrev, consistent with poor
stabilization of pHin. Taken together, these results indicate that the consistent change of Vrev to more
negative values is the result of cell acidification while bathed in
solutions of low pH.
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Fig. 4.
Measurement of pHin,
Vrev, and membrane conductance of an oocyte expressing the
M2 channel as it was exposed to a bathing solution of low
pH. A, time course of the changes. Note that
pHin (top graph) decreased after
Vrev (middle graph) reached its peak value while
conductance (lower graph) increased steadily during exposure
to low pH bathing medium. B, plot of Vrev
against pHin for each of the times shown (in seconds) after
lowering pHout from pH 8.5 to pH 5.8. The relationship
displayed three phases as follows: (i) from 0 to 20 s, immediately
after pHout was reduced, Vrev reached a peak
value, whereas pHin changed very little; (ii) from 20 to
120 s when Vrev returned to more negative values with
a small (~0.1 pH unit) change in pHin; and (iii) for
t >120 s when pHin changed slowly as
Vrev reached its plateau value.
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Fig. 5.
Measurement of pHin of CV-1 cells
that expressed the M2 protein using fluorescence of
BCECF. A, the pHin of cells loaded with
BCECF decreased when the pH of the control bathing medium ( 
, time
course in upper bar) was decreased from pH 7.4 to pH 6.2 and
returned to normal values when pH of the bathing medium was returned to
pH 7.4. When bathed in solution containing amantadine
(amant) (100 µM, 
, time course upper
bar) at pH 6.2 the pH did not decrease. Acidification did,
however, occur as a result of treatment with FCCP (
, time course
lower bar), following treatment with amantadine. Shown is
the average pH from four M2-expressing cells; error bars
were omitted for clarity; the largest S.E. was 0.12 pH unit.
B, changes of pHin for cells that were treated
with K+ ionophore valinomycin (valin) to
stabilize membrane voltage according to the membrane K+
gradient. Note that the acidification was more rapid for membrane
voltage of approximately 80 mV (3 mM K+
out, , time course in lower bar) than
for approximately 20 mV (60 mM K+
out, time course in upper bar), and
that lowering pH, in the presence of amantadine (amant) (100 µM, , time course upper bar), did not alter
pHin. Shown are average pH changes from seven
M2-expressing cells; error bars are omitted for
clarity.
The effect of Na+ replacement on M2 ion channel
activity
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Fig. 6.
Effect of replacement of Na+ in
the bathing medium with Li+ (A) or
NH4+ (B) on the
current-voltage relationship of oocytes expressing M2
protein. Note the decrease in conductance for the Li+
substitution and the increase in both conductance and Vrev
for the NH4+ substitution. Measurements
were made 20 s after changing solutions, at the time when
Vrev reached its peak value (see Fig. 1).
120 mV from a
holding voltage of 20 mV at pH 5.8 in the presence of low (0.15 mM) buffer concentration in the bathing medium. We measured
the diminution in current amplitude during the pulse and the final
current amplitude at the end of the pulse in low buffer,
Na+-containing medium. We then measured these variables in
low buffer media in which Na+ was replaced by
Li+ or NH4+, and we compared
the values. Replacement of Na+ by Li+ in a
solution containing 0.15 mM buffer caused a decrease in the
steady-state current amplitude, measured at the end of the hyperpolarizing pulse (to 64 ± 2.7% S.E., n = 10). We found that the diminution of current amplitude during the 2-s
voltage pulse was reduced proportionally (to 63 ± 4.5% S.E.,
n = 10) in a medium of low buffer concentration (Fig.
7). This result is consistent with Li+ interfering with
proton conduction through the M2 ion channel.
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Fig. 7.
Effect of reducing concentration of the
buffer in the bathing solution on the currents of oocytes expressing
the M2 protein that were bathed in media containing
Na+, Li+, and
NH4+. The decrease of
current amplitude during the 2-s-long pulse serves as a measure of
proton current, independent of the current of other ions, as the decay
only occurs when the buffer capacity of the outside solution is
lowered. Note that the reduction of current in 0.15 mM
buffer during a 2-s voltage clamp pulse from a holding potential of
20 to 120 mV was less for oocytes bathed in Li+ than
for oocytes bathed in Na+ and that the reduction in
amplitude persisted in NH4+-containing
solutions. Note also the smaller final current amplitude at the end of
the hyperpolarizing pulse in solutions containing Li+
compared with those containing Na+ or
NH4+.
Replacing Na+ with NH4+, on the other hand, caused an increase in the steady-state current amplitude in low buffer, measured at the end of the hyperpolarizing pulse (to 315 ± 20% S.E., n = 8). However, the buffering capacity of this solution, which contained 0.15 mM MES at pH 6.2, was unavoidably increased by the presence of 88 mM ammonium buffer (to 0.66 mM total buffering capacity at pH 5.8). The decrement of current amplitude during the voltage pulse was reduced to 61 ± 5.5% S.E. (n = 8) of that observed in Na+-containing low buffer (0.15 mM) solutions (Fig. 7), an effect that could be attributed to the modest increase of buffering capacity imparted by the 88 mM NH4+Cl solution (Mould et al. (23)). However, the large increase in final current amplitude in the NH4+-containing solution cannot be explained by this modest increase in buffer capacity and demonstrates that an additional current flows which is not affected by external buffer concentration. Similar results were obtained when Na+ was replaced with NH3OH+ (data not shown). Finally, replacement of Na+ with NMDG+ and NaCl with mannitol had no detectable effect on either the final current amplitude at the end of the pulse or the diminution of current during voltage pulses in low external buffer concentration (data not shown).
Comparison of M2-specific Oocyte Acidification with That Obtained from "Pure" Proton Currents Using an Electrogenic Protonophore
A final way that we tested the ion selectivity of the M2 ion channel was by comparing the acidification rate, normalized to the amplitude of the inward current, of M2-expressing cells bathed in solutions of low pH with that obtained in cells exposed to the electrogenic protonophore FCCP (30). If the M2 channel conducts only protons, then for a given inward current amplitude the acidification rate of an oocyte expressing the M2 protein at low pH should be equal to that observed in the presence of FCCP.
Oocytes Treated with FCCP--
Membrane current, Vrev,
and pHin over time were measured in uninjected oocytes
bathed in pH 5.8 Barth's solution in the presence or absence of 20 µM FCCP (in 0.02% Me2SO carrier; exposure to carrier alone produced no membrane currents). Oocytes clamped at a
holding voltage of 20 mV developed an inward current at low pH after
FCCP was added to the bathing medium (Fig.
8A, upper record). The
amplitude of the inward current of FCCP-treated cells was generally
less than that of M2-expressing cells studied at the same
pH (compare upper and lower records of Fig.
8A), but it was found that applying higher concentrations of
FCCP resulted in deterioration of the condition of the cells. The
inward current normally appeared within 1 min of exposing cells to
FCCP, a delay that was probably due to the time required for
incorporation of FCCP into the oocyte plasma membrane. Vrev
measured using voltage ramps shifted within a few seconds to positive
potentials after the pH of the bathing medium was reduced (Fig.
8B, lower record). As observed for M2-expressing
oocytes held at 20 mV, the Vrev of oocytes exposed to
FCCP (70.4 ± 1.6 mV S.E., n = 10) was close to
EH+ predicted from the known pHout
and pHin measured with an intracellular pH electrode
(81.8 ± 5.2 mV S.E., n = 10). After reaching a
maximum, Vrev began to return to more negative potentials, and the current began to decrease in amplitude a few minutes after the
pH of the bathing medium was decreased (Fig. 8). The pHin of oocytes treated with FCCP did not begin to decrease until about 100 s after the changes in current and Vrev occurred
(Fig. 8B, upper record). One important difference was noted
between the behavior of oocytes expressing the M2 protein
and those into which FCCP had been incorporated. When the pH of the
bathing solution of FCCP-treated oocytes was returned to pH 8.5 following oocyte acidification, a large, transient outward current
appeared (Fig. 8A, upper record). The appearance of this
transient outward current was accompanied by an overshoot of
Vrev to potentials more negative than those observed before
lowering pH of the bathing medium (Fig. 8B, lower
record). This was in contrast to the measurements of M2-expressing oocytes for which no outward current flowed
upon return to bathing medium of pH 8.5 after prolonged bathing in low
pH medium (Fig. 8A, lower record). The incorporation of FCCP into the plasma membrane appeared to be reversible, as re-exposure of
oocytes to medium of pH 5.8 following washout of FCCP in medium of pH
8.5 failed to produce an inward current (data not shown).
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The maximal rate of acidification, normalized to the maximum inward current amplitude, of M2-expressing oocytes was compared with the same value obtained from uninjected oocytes treated with the FCCP ionophore. It was found that the ratio of maximal rate of acidification to maximal inward current to be similar in both cases (0.085 ± 0.006 pH unit/min/µA for FCCP-treated cells, n = 8, versus 0.081 ± 0.11 pH unit/min/µA for M2-expressing cells, n = 20).
CV-1 Cells Treated with FCCP--
We performed two types of
experiments. In the first type of experiment CV-1 cells that did not
express the M2 protein were employed, and membrane currents
were measured at low pHout in the presence of FCCP (Fig.
9). In the second type of experiment, we
studied the effect of FCCP treatment on the pHin of
M2-expressing cells after inhibiting M2
currents with amantadine (Fig. 5A). The results of both
types of experiments were similar to those obtained with oocytes.
Lowering the pH of FCCP-treated cells that did not express the
M2 protein from pH 7.4 to pH 6.2 resulted in an inward
current flow accompanied by an initial increase in Vrev to
positive voltages and an increase in conductance (Fig. 9). If the
holding voltage was adjusted to give very little inward current while
the cells were bathed in low pH medium, the measured Vrev
was 65.8 ± 3.5 mV S.E. (n = 6). This value was
close to the 69.6 mV value of EH+ calculated
from the known pHout and an assumed pHin of pH
7.4. However, when the holding voltage was made more negative, the
Vrev quickly returned to more negative voltages (Fig.
9).
|
The effect of FCCP on pHin was also measured in CV-1 cells
(Fig. 5A). In this experiment, M2-expressing
cells were exposed first to pH 6.2 for ~200 s and then allowed to
recover from acidification in a bathing medium of pH 7.4. The cells
were then reexposed to pH 6.2 in the presence of 100 µM
amantadine, and in this solution acidification did not occur. Finally,
the same cells were exposed to pH 6.2 medium in the presence of FCCP
for 200 s. This treatment resulted in acidification once again.
Finally, the cells were allowed to recover from acidification in medium
of pH 7.4 + FCCP. Bathing FCCP-treated cells in solution of pH 6.2 resulted in an acidification that occurred with a slightly more rapid
time course than that observed for M2-expressing cells
(Fig. 5A). The pHin of the
M2-expressing cells was pH 6.96 ± 0.15 pH units S.E.
(n = 5) at pH 7.4out before application of
FCCP, and pHin decreased by 0.88 ± 0.095 pH units
(n = 5) after reducing pHout from pH 7.4 to
pH 6.2 in the presence of FCCP. As also observed with oocytes, these
changes of pHin were reversible upon return to bathing
medium of pH 7.4. It was not possible to study CV-1 cells in solutions of pH 8.5 because irreversible changes occurred at this alkaline pH
value. These two experiments demonstrate that acidification of CV-1
cells can also be achieved by treatment with FCCP and that
acidification results from a mechanism that is not affected by the
presence of the M2 protein.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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This study confirms the very high proton selectivity of the M2 ion channel under physiological conditions, demonstrates that Li+ inhibits the channel, and provides additional evidence that the channel allows the permeation of quaternary ammonium ions, probably by a different mechanism than that for H+. These results also provide evidence for the restricted diffusion of H+ in the cytoplasm of oocytes and support the notion that the M2 channel is gated by changes in pHout.
These results are consistent with the proposed roles for M2
ion channel in the life cycle of the influenza A virus. However, the
only known ion channel encoded by influenza A virion is the M2 ion channel, and a pure proton conductance for the
M2 channel would depolarize the virion membrane. This
depolarization would elevate virion membrane potentials to high enough
values to risk dielectric breakdown of the membrane and decrease the
driving force on protons. Thus, this depolarization would limit the
extent of virion acidification that is possible. A simple calculation can estimate the number of protons that could flow into the virion without causing an excessive virion membrane potential that would lead
to dielectric breakdown of its lipid bilayer. If we assume that the
virion starts with a membrane potential of about 70 mV (the resting
potential of many epithelial cells) and assume that it can withstand a
membrane voltage of +100 mV, then about ~2.5 × 1022 Eq of protons will be able to enter the
virion during acidification, changing its membrane voltage by altering
the charge across its membrane capacitance, before placing the virion
membrane in danger of dielectric breakdown. Assuming that the volume of
the virion is about 1.5 × 1019 liters
and that the interior of the virion is well buffered, the pH inside the
virion will decrease by only a few tenths of a pH unit if the pH
decrease is distributed uniformly throughout the interior of the
virion. The reason that the calculated pH decrease is so small is that
every proton that enters the virion carries a charge that increases the
membrane voltage of the virion, but only a small fraction of the
entering protons contribute to the free proton concentration (lower pH)
because the buffering capacity of the virion proteins can sequester the
majority of the protons. Thus, the calculated decrease of pH of the
virion, if it occurred uniformly throughout the virion, would only
lower pH of the virion from an assumed initial value of approximately pH 7.4 to a value slightly higher than pH 7.0. This decrease of pH is
inadequate to cause release of ribonucleoprotein complexes from the M1
matrix protein (31) contained within the entire virion. Perhaps the
acidification of only a zone of the virion immediately below the virion
surface membrane might be adequate (32, 33), as this is the zone where
the M1 protein is concentrated. It is intriguing that we found evidence
for a localized pH decrease in the zone near the cytoplasmic entrance
to the pore of the M2 channel in oocytes.
The Difficulty of Determining M2 Ion Selectivity by
Comparing Vrev with EH+ Calculated from
Measured pHin--
In a previous study Shimbo et
al. (12) found that when M2-expressing oocytes were
bathed in a solution of low pH, Vrev was 20-30 mV more
negative than the value predicted from EH+ calculated from the pHin measured with a micro pH electrode
and the pH of the bathing solution. In that study, Vrev
measurements were made ~2 min after the oocytes were exposed to
bathing solutions of low pH, at approximately the time when the
amplitude of the inward membrane current reached a maximum value, to
ensure adequate equilibration of the low pH solution. The relative
permeability of H+ to that of Na+ was found to
be about 105. In the present study it was found that upon
lowering extracellular pH, Vrev measured in
M2-expressing oocytes shifted within a few seconds toward
positive potentials near EH+ and then fell to
more negative values before inward current reached its maximum amplitude (Fig. 1B, oocytes). Similar results were obtained
in CV-1 cells that expressed the M2 protein (Fig. 2, CV-1
cells). The return of Vrev to more negative values was not
the result of activating an endogenous current, as the shift still
occurred in CV-1 cells expressing the M2 protein in the
absence of other conducting ions. The return of Vrev to
negative values was also not due to inactivation of the M2
ion channel, as the slope conductance increased as Vrev
became more negative. This finding is consistent with results obtained
for HEK293 cells expressing the Kv2.1 delayed rectifier channel (34).
In HEK293 cells the activity of the channel produces changes in
[K+]in that mimic inactivation and results in
apparent changes in ion selectivity. By using the peak value of
Vrev and the pHin measured at the time of peak
Vrev, the permeability of H+ relative to that
of Na+ was found to be about 1.5-2.0 × 106, consistent with the values found by Chizhmakov and
co-workers (5) and consistent with results from reconstituted
M2 protein in vesicles (35, 36). For CV-1 cells studied in
the absence of Cl and K+ ions and two oocytes
studied in control solution, the measured values of Vrev
and the calculated values of EH+ were within a
few mV, consistent with a high proton permeability.
Two lines of evidence indicate that acidification of the cytoplasm of cells expressing the M2 ion channel is the explanation for the return of Vrev to more negative values, after reaching a peak positive value, when cells are bathed in medium of low pH. First, the rate of the return of Vrev was smaller when cells were held at voltages closer to EH+ (Fig. 3). Second, for times longer than 120 s after introduction of the low pH bathing solution, the change in Vrev and the change in pHin had a similar time course (Fig. 4).
Several observations indicate that H+ diffusion in oocytes is restricted by the presence of immobile buffers (37-39). First, the decrease in pHin occurred with a delay after the onset of inward current (Fig. 4). Second, the return of Vrev to negative values following the occurrence of its peak value near EH+ occurred prior to the change in pHin detected using an intracellular pH electrode (Fig. 4). The data presented here are also consistent with previous conclusions that it is very difficult to control the pHin of a cell when the diameter of the patch pipette is much smaller than that of the cell. Even with very high concentrations of buffers, the diameter of the patch pipette needs to be no less than one-third of the cell diameter in order to control pHin adequately (28). As the CV-1 cells used in our patch clamp experiments were 100-150 µm diameter and the largest pipettes we were able to use were 3-4 µm diameter, it would be expected that pHin was not well controlled in our experiments, even when high concentrations of buffer were used. Chizhmakov and co-workers (5) found that the Vrev of M2-expressing MEL cells was close to the value predicted