From the Department of Physiology, Osaka City University Medical School, Abeno-ku, Osaka 545, Japan
Proton (H+) conductive pathways are suggested to play roles in the regulation of intracellular pH.
We characterized temperature-sensitive whole cell currents in mouse bone marrow-derived mast cells (BMMC), immature proliferating mast cells generated by in vitro culture. Heating from 24 to 36°C reversibly and repeatedly
activated a voltage-dependent outward conductance with Q10 of 9.9 ± 3.1 (mean ± SD) (n = 6). Either a decrease
in intracellular pH or an increase in extracellular pH enhanced the amplitude and shifted the activation voltage
to more negative potentials. With acidic intracellular solutions (pH 5.5), the outward current was detected in
some cells at 24°C and Q10 was 6.0 ± 2.6 (n = 9). The reversal potential was unaffected by changes in concentrations of major ionic constituents (K+, Cl, and Na+), but depended on the pH gradient, suggesting that H+
(equivalents) is a major ion species carrying the current. The H+ current was featured by slow activation kinetics
upon membrane depolarization, and the activation time course was accelerated by increases in depolarization, elevating temperature and extracellular alkalization. The current was recorded even when ATP was removed from
the intracellular solution, but the mean amplitude was smaller than that in the presence of ATP. The H+ current
was reversibly inhibited by Zn2+ but not by bafilomycin A1, an inhibitor for a vacuolar type H+-ATPase. Macroscopic measurements of pH using a fluorescent dye (BCECF) revealed that a rapid recovery of intracellular pH
from acid-load was attenuated by lowering temperature, addition of Zn2+, and depletion of extracellular K+, but
not by bafilomycin A1. These results suggest that the H+ conductive pathway contributes to intracellular pH homeostasis of BMMC and that the high activation energy may be involved in enhancement of the H+ conductance.
The importance of intracellular pH (pHi)1 regulation
is unquestionable in various cellular functions, and the
pHi regulatory mechanisms vary considerably among
different types of cells (Hoffmann and Simonsen, 1989).
Recently voltage-activated H+ currents were found in
certain cell types of hematopoietic (DeCoursey and
Cherny, 1993
; Demaurex et al., 1993
; Kapus et al., 1993
;
Holevinsky et al., 1994
; Nordström et al., 1995
) and
nonhematopoietic origin (Thomas and Meech, 1982
;
Byerly et al., 1984
; DeCoursey, 1991
; Krause et al.,
1993
). The H+ conductive pathway is considered to
contribute significantly to pHi homeostasis, as well as ion
exchangers, H+ pumps and ion cotransporters. However,
permeation mechanisms of the H+ conductance remain to be proved, and the putative H+ channel is suggested to be different from the general water-filled ion
channels (DeCoursey and Cherny, 1995
).
Bone marrow-derived mast cells (BMMC) are immature, proliferating mast cells differentiated from multipotential hematopoietic stem cells of bone marrow by
in vitro culture in the presence of mast cell growth factors (Ihle et al., 1983; Razin et al., 1984
). These cells
have characteristics of mast cells, secreting histamine
and other chemical mediators upon antigenic stimulation, and also have the potency to differentiate into the
different peripheral phenotypes (Nakano et al., 1985
).
BMMC offer an excellent model to study roles of ion
channels in different functional and developmental
states. We recently reported that electrophysiological properties of BMMC were heterogeneous (Kuno et al.,
1995a
) but could not identify H+ conductance. These
recordings were made at room temperature, however,
and a functional significance of the membrane conductances should be evaluated at physiological temperature.
The present data provide evidence for the presence
of a H+ conductive pathway in BMMC. The H+ conductance shares common electrophysiological features with those in other cell types (voltage- and time-dependent
activation, sensitivity to both pHi and pHo and blockage
by heavy metals) (Byerly et al., 1984; DeCoursey and
Cherny, 1994
) but is distinct in its high sensitivity to
temperature. The presence of a temperature-sensitive and voltage-dependent H+ efflux is confirmed by measuring pHi using BCECF. The mean conductance is reduced by omitting intracellular ATP, so that ATP may
modulate the current activity at least partly. The H+
conductive pathway would contribute to pH regulation
of BMMC during metabolic acidosis associated with cell
growth, production of large amounts of chemical mediators, or performance of other cellular functions. A
preliminary account has been made (Kuno et al.,
1995b
).
Cells
Bone marrow cells were obtained from the femoral and tibial
bones of male, 8-10-wk-old mice (Balb/ca). The mice were killed by an overdose of ether. Pokeweed mitogen-stimulated spleen
cell-conditioned medium (PWM-SCM) was prepared as described elsewhere (Nakahata et al., 1982). Briefly, mouse spleen
cells were incubated at 37°C in a 95% air-5% CO2 atmosphere
with pokeweed mitogen (1:300 dilution) (GIBCO BRL Products,
Gaithersburg, MD) in
-MEM (INC Pharmaceuticals Inc., Irvine,
CA) supplemented with 10% FCS (INC Pharmaceuticals Inc.),
10
4 M 2-mercaptoethanol, streptomycin (0.1 mg/ml), penicillin
(100 U/ml), and amphotericin B (0.25 µg/ml). After 5 d of incubation, the supernatant (PWM-SCM) was filtered with a millipore
filter (0.22 µm) and stored at
85°C. Bone marrow cells were
plated at 106/ml in 35-mm petri dishes and were incubated at
37°C in a 95% air-5% CO2 mixture. The culture medium contained
-MEM, 10
4 M 2-mercaptoethanol, 10% FCS, and 10%
PWM-SCM. Half of the medium was changed every week. Cells
densely stained with alcian blue (Worthington, 1962
) were identified as BMMC. BMMC appeared within 2-4 wk and were maintained for more than 6 months. Cells cultured for more than 4 wk were used in this experiment, because the purity of BMMC
was
90-95%.
Solutions
The standard pipette solution contained (mM): 150 K-glutamate,
7 MgCl2, 1 EGTA, 1-2 Na2ATP, and 10 HEPES (pH = 7.3). In
some experiments, K-glutamate was replaced by CsCl. In acidic
intracellular solutions, pH was buffered with 10 or 120 mM Mes.
With 120 mM Mes, K-glutamate or CsCl was reduced to 100 mM
to compensate for the osmolality, and the pH was adjusted to 5.5 by 14-20 mM KOH. The pH of the pipette solutions was designated as pHp. The standard extracellular solution contained: 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES,
10 mM glucose, and 0.1% BSA (pH = 7.3). Extracellular solutions were buffered with 10 mM Mes for pH 6.7 and with 10 mM 2-(cyclohexylamino)ethanesulfonic acid (CHES) for pH
8.2. NaCl was replaced by KCl to make a K+-rich (150 mM) solution and by N-methyl-D-glucamine (150 mM) to make a NMG+-rich solution. A low Cl
solution was prepared by replacing NaCl
with Na-isethionate. With 10 mM buffers, the pH was adjusted by
KOH (3-8 mM) in the intracellular or K+-rich external solutions
and by NaOH (3-8 mM) in other external solutions.
Recordings
Current signals were recorded from the whole-cell voltage clamp
configuration. The reference electrode was an Ag-AgCl wire connected to the bath solution through a Ringer-agar bridge. Pipette resistance ranged between 5 and 15 M. The zero-current potential before formation of the gigaseal was taken as 0 mV. The recording chamber was placed on a heating plate (MT1; Narishige,
Tokyo, Japan), and the bath temperature was changed between
24 and 36°C. The current signals were led into a patch-clamp amplifier (EPC7; LIST, Darmstadt, Germany) without series resistance
compensation, digitized at 1-4 kHz with an analog-digital converter (MacLab/4; Analogue Digital Instruments, Australia) and
stored on a personal computer. Cell capacitance was estimated
with capacitive cancellation circuitry on the amplifier (5.3 ± 1.0 pF, n = 142). We started to collect data within 1-2 min after the
formation of the whole cell configuration, since the H+ current
appeared within 10-30 s after the rupture of the patch membrane and usually did not further increase at 32-36°C. Leak current was determined from the linear portion of the current-voltage (I-V) relation when either inward or outward current was absent or when the currents were eliminated by the blockers. The
inward and outward conductances were obtained from the I-V relation between
110 and
80 mV and between +50 and +100
mV, respectively, after subtraction of the leak current. To obtain
the I-V relation, voltage pulses were applied at an interval of 3 min. All data were expressed as mean ± SD. Statistical significance was evaluated with Student's unpaired t test. The goodness
of fit of single exponential distribution to the activation kinetics
and tail currents was determined by the
2 test. In most cases, P
values for the fit were greater than 0.95.
Measurements of pHi by BCECF
Macroscopic pHi was determined by loading populations of
BMMC (3-6 × 105 cells/ml) with a pH-sensitive fluorescent dye,
2,7
-bis-(2-carboxyethyl)-5 (and -6) carboxyfluorescein (BCECF).
Cells were incubated with the acetoxymethyl ester form of
BCECF (BCECF-AM) (1 µM) for 30 min and then, after washing
the dye, with 40 mM NH4Cl for 15 min at 36°C. Washing cells by
ammonium-free solutions rapidly acidified cells (Roos and Boron, 1981
). Recovery of pHi in the acid-loaded cells was measured by a spectrofluorometer (650-60; Hitachi, Tokyo, Japan).
The ratio of fluorescences excited at two wavelengths, 490 and
450 nm (bandwidth; 3-5 nm), was obtained at an interval of 1 min. The emission wavelength was 530 nm (bandwidth; 6-10 nm). Calibration of pHi was carried out by dissipating plasma membrane pH gradient with 10 µM nigericin in a K+-rich solution with known pH values (Thomas et al., 1979
; Grinstein and
Furuya, 1988
). To eliminate contamination with nigericin (Richmond and Vaughan-Jones, 1993
), the cuvette and teflon-coated
spin bar were rinsed by ethanol before use. The rate of the pHi
recovery was estimated by differentiating the pHi recordings.
Substances
A condensed stock solution of Na2ATP (500 mM) was prepared
in 1 M Tris Cl, stored in a freezer, and added to the internal medium before use. Condensed stock solutions of 4,4-diisothiocyano-2,2
-stilbenedisulphonate (DIDS) and ZnCl2 were dissolved
in distilled water. The free concentration of Zn2+ was not determined and the concentration described herein is only nominal,
since Zn2+ forms complexes with anions. Bafilomycin A1 was prepared in ethanol. The final concentration of ethanol was
0.5%.
BCECF-AM, CHES and Mes were obtained from Dojindo Laboratories (Kumamoto, Japan), and other chemicals were obtained
from Sigma Chemical Co. (St. Louis, MO).
Elevation of Temperature Activates an Outward Current
Fig. 1 A illustrates a representative time course of changes
in the whole cell current when temperature of the recording chamber was altered (top). Upward and downward vertical lines in the bottom trace represent outward and inward currents evoked by voltage ramps
(200 mV/s) applied at a holding potential of 0 mV
(middle). Heating from 24 to 36°C reversibly and repeatedly activated outward currents both in the K+-rich
(150 mM K+) and the standard Ringer (5 mM K+) solutions. Fig. 1, B and C, show superimposed current-voltage (I-V) relations obtained by the voltage ramps during heating in the K+-rich (B) and the standard Ringer
(C) solutions.
Temperature-conductance relationships (see MATERIALS AND METHODS) in three cells show that heating
from 24 to 36°C gradually increased the outward conductance, although the magnitude varied greatly among
cells (Fig. 2 A). The inward conductance of these cells
remained unchanged or increased only slightly by the
heating (Fig. 2 B). Fig. 2 C summarizes semilogarithmic
plots of the outward conductance against temperature
in six cells with a linear regression for all data. The conductance was normalized to that measured at 36°C. The
factor by which the conductance changes per 10°C elevation (Q10) was 9.9 ± 3.1 (n = 6).
Although the outward current described above was recorded at pHp (pH of the pipette solution) 7.3, the current larger than 10 pA at the end of a 500-ms voltage pulse of +100 mV was evident only in 7 of 19 cells at 32°C. The current was seen more frequently at lower pHp. At pHp 5.5, 80 of 103 cells exhibited the outward current, although the percentage of cells with the current varied among different cell batches from 70 to 92%. The magnitude normalized by cell capacitance was generally much smaller at pHp 7.3 (2.4 ± 7.3 pA/ pF, n = 19) than that at pHp 5.5 (9.6 ± 9.5 pA/pF, n = 103). At pHp 5.5, the outward current was detected in some cells even at 24°C and was enhanced by heating with Q10 of 6.0 ± 2.6 (n = 9). In later experiments, the heating-activated outward current was analyzed at pHp 5.5 to optimize the detection of the current unless described otherwise.
Fig. 3 A shows a family of currents activated by depolarization pulses in +20-mV increments applied at 40
mV at 32°C. The outward current was characterized by
slow activation kinetics at depolarization, differing from
the rapidly activating, outwardly rectifying Cl
current
that we reported previously (Kuno et al., 1995a
). Activation followed a single exponential time course (thin lines)
after a small delay and was facilitated at more positive
potentials. The activation time course was also greatly
affected by temperature. Fig. 3 B illustrates superimposed outward currents evoked by a depolarizing pulse
to +100 mV applied at
40 mV when temperature was
elevated from 24 to 34°C in a cell. Relationships between the activation time constant and the membrane
potential at different temperatures in this cell indicate
that at any given potential the time constant becomes
shorter at higher temperatures (Fig. 3 C). Similar results were obtained from five cells tested. Further experiments were performed at 32°C, as recordings at
36°C were often unstable.
The Heating-activated Outward Current Is Mediated Mainly by Proton (H+)
Averaged I-V plots obtained from different cells at
three pHp (5.5-7.3) are presented in Fig. 4 A. The pHo
was the same (7.3) in all cases. The currents were measured at the end of 500-ms voltage pulses. The acidic intracellular solutions were buffered either with 10 or
120 mM Mes. Little current was detectable at potentials
more negative than 0 mV. Although there was cell-to-cell variability in the potential at which activation occurred, lowering pHp shifted the voltage dependency
to a more negative direction. On the other hand, elevating pHo from 7.3 to 8.7 reversibly shifted the I-V curves
to a more negative direction in all cells tested (n = 12)
(Fig. 4 B). This shift by extracellular alkalization was
observed even in cells recorded with the pipette containing CsCl (Fig. 4 C). The time course of activation
was accelerated by increasing pHo. At +80 mV with pHp
5.5, the time constant at pHo 8.2-8.7 was 327 ± 61 ms
(n = 5), significantly faster than that at pHo 7.3 (625 ± 255 ms, n = 11; P < 0.05).
The outward currents appeared within 10-30 s after
the rupture of the patch membrane to form the whole
cell configuration with the acidic pipette solutions buffered by either 10 or 120 mM Mes. Thus BMMC seemed
to be acidified quickly, probably because of the small
cell size (radius; 5.1 ± 0.7 µm, n = 142). The current amplitude at the end of a 500-ms voltage pulse of +100
mV at pHo 5.5 was 8.9 ± 10.0 pA/pF (n = 63) with 10 mM Mes, not significantly different from that with 120 mM Mes (10.8 ± 8.7 pA/pF, n = 40). I-V relations obtained by 500-ms voltage pulses from 100 to +100 mV, and intermittent voltage ramps were not affected
by the buffering power. However, when a 1-s depolarizing pulse (+100 mV) was repetitively applied at an interval of 3 s, a gradual rundown of the outward current
was observed with 10 mM Mes (by 19.1 ± 18.6% per 10 pulses, n = 16) but not with 120 mM Mes (by
1.5 ± 11.0% per 10 pulses, n = 14). In addition, when the
cells were exposed to an 8-s depolarization (+60 mV),
the current amplitude gradually increased to a peak and then reduced to 87.0 ± 9.3% (n = 11) of the maximum at the end of the depolarization with 10 mM Mes
and 95.2 ± 5.6% (n = 11) with 120 mM Mes. The decline was greater with the lower concentration of Mes
(P < 0.05). Thus a high buffering power in pipette solutions was needed to maintain the current.
To determine ion species carrying the current, the
currents were studied under various experimental conditions. The outward current was observed both when
the extracellular Cl ([Cl
]o) was totally replaced by
isethionate
(Fig. 5 A) and when the intracellular K+
was totally replaced by Cs+ (Fig. 5 B). Tail currents at 0 mV after the depolarizations were directed outwardly
(arrow) and were fit by single exponential curves as indicated by superimposed thin lines in the magnified record (right). In these recordings, the external medium contained a Cl
channel blocker, DIDS (50 µM),
which blocks the Cl
current in BMMC (Kuno et al.,
1995a
). Addition of a K+ channel blocker, Ba2+ (1 mM)
did not affect the current. When the pHo was 6.0, the outward current was small in most cells, but sizable currents were observed in a few cells (Fig. 5 C). Tail currents in these cells were going inwardly at 0 mV, suggesting that lowering pHo from 7.3 to 6.0 shifted the reversal potential (Vrev) to a more positive level.
The Vrev was estimated from the I-V plots for instantaneous tail currents recorded in cells with larger currents. The voltage protocols were made of test pulses after the 1-s prepulse (either +80 or +100 mV) (Fig. 6
A). The amplitudes of tail currents at the start of each
test pulse were determined from the single exponential fit as shown in Fig. 5. Closed circles in Fig. 6 B represent the averaged Vrev at pHp 5.5 recorded with 120 mM
Mes in the pipette solutions. The Vrev became more
negative in alkaline media. The dashed line represents
a linear regression for data at pHo 6.0-7.3 with 120 mM
Mes, having a slope of 45.5 mV per pHo of 1. Data at
higher pHo deviated from the regression line. Departures from the line were more prominent with 10 mM
Mes (open circles). When the Vrev was replotted against
the ratio between extracellular and intracellular Cl
concentrations ([Cl
]o and [Cl
]i) on a semilogarithmic scale (Fig. 6 C), the Vrev did not depend on the Cl
gradient. Open and closed squares represent data recorded with pipette containing K-glutamate and CsCl,
respectively, showing that the Vrev did not depend on
the K+ concentration. These results suggest that the
heating-activated outward current is carried primarily
by H+ (equivalents).
Effects of Zinc, Bafilomycin A1, and ATP on the H+ Current
A blocker for H+ conductances, ZnCl2 (0.25-0.5 mM),
reversibly suppressed the outward current to 10 ± 10.3% (n = 8) of the controls (Fig. 7). On the other
hand, the current amplitude in the presence of bafilomycin A1 (100 nM), a potent and selective inhibitor for
the H+-ATPase (Bowman et al., 1988), was 47.1 ± 37.5 pA at the end of a 500-ms pulse of 100 mV (n = 20),
not significantly different from the controls (56.2 ± 41.0 pA; n = 22). It seems that the H+ current is not
mediated via an electrogenic vacuolar type H+-ATPase.
As high sensitivity to temperature implicated that a
high activation energy would be involved in the current
activity, we examined the effects of intracellular ATP
on the H+ current. Fig. 8 shows I-V plots from cells recorded with ATP-containing (A) and ATP-omitting (B)
pipette solutions. The data were obtained from the same
batch of cells, to avoid batch-to-batch variation. The H+
conductance recorded without ATP was 104 ± 54 pS/pF
(n = 10), significantly smaller than that observed with
ATP (216 ± 143 pS/pF; n = 10) (P < 0.05). Thus, ATP
might be involved in potentiation of the H+ current.
Measurements of Macroscopic pHi Recovery in Acid-loaded Cells
To assess the role of the H+ conductance in regulation
of pHi, pHi recovery from acid-load in populations of
BMMC was measured using a fluorescent pH-sensitive
dye, BCECF (Fig. 9). Washing cells preincubated with
NH4Cl by ammonium-free solutions induced intracellular acidification (Roos and Boron, 1981), followed by a
pHi recovery in an Na+-free K+-rich solution at 36°C
(Fig. 9, closed circles) (see MATERIALS AND METHODS). Although the time course and the resultant pH level at
~20 min differed among preparations, the pHi recovery was characterized by an initial rapid phase within
5-10 min. The Na+-independent pHi recovery was reduced at 24°C (Fig. 9 A). In an Na+-free NMG+-rich
medium (Fig. 9 B), which would hyperpolarize cells,
the pHi recovery was greatly inhibited, suggesting that
depolarization is required for the rapid H+ efflux. Addition of 0.5 mM ZnCl2 attenuated the pHi recovery (Fig. 9
C), but bafilomycin A1 (100 nM) did not (Fig. 9 D).
The inhibitory effect of low temperature and Zn2+
seemed to be more prominent during the initial phase
of pHi recovery. The rate of the pHi change per time
(pH/min), obtained by differentiating the pHi recordings, was attenuated by lowering temperature
(open circles) and was greatly inhibited by 0.5 mM
Zn2+(squares) (Fig. 10). The rate at 0 min was 0.049 ± 0.029 pH U/min (n = 7) at 24°C, 0.010 ± 0.010 pH
U/min (n = 5) in the presence of Zn2+, and 0.110 ± 0.068 pH U/min (n = 12) in their controls. These results support an idea that a temperature- and Zn2+-sensitive H+ conductive pathway is responsible for a rapid
pHi regulation during intracellular acidification. The
slow pHi recovery remaining in the presence of Zn2+
may be mediated by unidentified H+ efflux mechanisms other than the H+ conductance.
A Temperature-sensitive H+ Conductance
We recently reported that an electrophysiological profile of BMMC was heterogeneous at room temperature,
such that an inwardly rectifying K+ current and an outwardly rectifying Cl current were exhibited in subpopulations (Kuno et al., 1995a
). The present study described a voltage-activated outward current which was
negligible or small at room temperature but was remarkably augmented by heating up to 36°C. H+ (equivalents) was suggested to be an ion species primarily responsible for the heating-activated current from several
lines of evidence. First, either a decrease in pHp or an
increase in pHo increased the current amplitude and
shifted the activation voltage to more negative potentials. Second, the current was observed even when the
extracellular Cl
was replaced by an impermeable anion, isethionate
, and when the intracellular K+ was replaced by Cs+. Third, the Vrev was dependent on the H+
gradient but was unaffected by the substitution of other
major ion constituents (Cl
, K+, Na+). Fourth, the current was not inhibited by K+ or Cl
channel blockers
(Ba2+ and DIDS) but by Zn2+, a blocker for voltage-activated H+ currents (Kapus et al., 1993
).
The H+ current in BMMC shares major properties of
that in other cell types (voltage- and time-dependent
activation, outward rectification, sensitivity to both pHo
and pHi, and a blockage by heavy metals) but is distinct
in its high sensitivity to temperature. Although H+ currents of snail neurons have a stronger temperature sensitivity than K+ currents over a temperature range of
10-25°C (Byerly and Suen, 1989), sizable H+ currents
were described in many mammalian cells at room temperature. It is possible that the H+ conductive pathway
comprises a diverse family of related types whose characteristics may be tissue specific and that activation mechanisms of the H+ current in BMMC may differ
from that in other cells. However, literature on the sensitivity of voltage-activated H+ currents to temperature
is too scarce to draw this conclusion at this moment.
Characteristics of the H+ Current in BMMC
Although H+ (equivalents) was estimated to be a primary constituent for the heating-activated outward current in BMMC, the Vrev deviated from the EH predicted
by the Nernst formula especially at high pHo, even
when the current was recorded with a high buffering power (120 mM Mes) in the pipette solutions. Prepulse
protocols consisted of different durations (0.5-4 s),
and the holding potentials (0 and 60 mV) did not affect the result. Similar deviation of the Vrev was often
described with H+ currents in other cell types (Mahaut-Smith, 1989
; DeCoursey, 1991
; Bernheim et al., 1993
;
Demaurex et al., 1993
; Kapus et al., 1993
), and some intrinsic buffering action or diffusion limitation suggested a deviation from the nominal pHp value (DeCoursey, 1991
; Kapus et al., 1993
; Holevinsky et al.,
1994
). The deviation of the Vrev from the expected
value was greater with 10 mM Mes, although the buffering power of the pipette solutions (10 or 120 mM Mes) did not significantly affect the current amplitude obtained by 500-ms step pulses or ramp pulses from
100
to +100 mV. This may result from depletion of the intracellular H+ concentration during repetitive prepulses.
Decline of voltage-activated H+ currents during long-lasting depolarization has been reported (Barish and
Baud, 1984
; DeCoursey, 1991
; Demaurex et al., 1993
; Kapus et al., 1993
) and is explained by depletion of H+
currents caused by preceding H+ efflux (Kapus et al.,
1993
). It seems that a high intracellular buffering
power is needed to maintain the H+ current during
long-lasting or repetitive depolarization.
So far, direct electrophysiological evidence that the
H+ currents are mediated by channels is not available
(Lukacs et al., 1993), as the current noise is small and
single channel current is hardly seen even in isolated
patches (DeCoursey and Cherny, 1995
). Noise analysis
estimated that the single channel conductance is <10
fS (Byerly and Suen, 1989
; Bernheim et al., 1993
; DeCoursey and Cherny, 1993
). In isolated patches of alveolar epithelial cells, H+ permeation mechanisms through
voltage-activated H+ channels suggest a difference
from those permeated through water-filled ion channels (DeCoursey and Cherny, 1995
). The current noise
was small and was not augmented by depolarizations in
BMMC as well. Little noise, high sensitivity to temperature, and a slow activation rate raises a possibility that
H+-conducting molecules in BMMC may be intermediate between channel and carrier proteins, although
this is only conjecture.
Effects of ATP on the H+ Current
A vacuolar type H+-ATPase is known as an electrogenic
H+ transport system. The H+-ATPase, generally localized in membranes of intracellular organelles, is found
in the plasma membrane of osteoclasts (Väänänen et al., 1990) and macrophages (Swallow et al., 1988
). The
Fo component of the H+-ATPase in osteoclasts forms a
channel protein to secrete H+ (Junge, 1989
). As osteoclasts generate from bone marrow cells, it is conceivable that BMMC would possess a similar plasmalemmal H+-ATPase. However, the H+ current in BMMC cannot
be well explained by a vacuolar type H+-ATPase for the
following reasons: First, the current is recorded in
some cells even when ATP is omitted from the intracellular milieu. Second, bafilomycin A1, a selective and potent blocker for the vacuolar type H+-ATPase (Bowman
et al., 1988
), does not block the current. Third, immunohistochemical studies documented that the antibody
for the vacuolar H+-ATPase labels osteoclasts but not
other cell types of bone marrow (Väänänen et al.,
1990
). Fourth, the ATPase can pump H+ against a
10,000-fold uphill concentration gradient (Heldrich et al.,
1989
), but only outward currents are activated at potentials positive to EH in BMMC. On this basis and together
with the electrophysiological properties (steep voltage-dependence, gating kinetics, the Vrev), the H+-ATPase
is unlikely to mediate the H+ current.
In many cell types, H+ currents are recorded in the
absence of ATP (Byerly and Suen, 1989; DeCoursey,
1991
; DeCoursey and Cherny, 1993
; Kapus et al., 1993
),
and a dependency on ATP has not been described. Although the H+ current was activated in some BMMC
even without ATP, the current amplitude was smaller
than that in the presence of ATP. Thus ATP may enhance the current activity either directly or indirectly: the H+ currents are reported to be amplified by cytoplasmic factors, such as arachidonate (Decoursey and Cherny,
1993), an elevation of intracellular Ca2+ (Holevinsky et
al., 1994
) or phorbol esters (Henderson et al., 1988
;
Nanda and Grinstein, 1991
; Kapus et al, 1992). A direct action of ATP on the molecule mediating the H+ conductance is an alternative explanation for the ATP dependency. The mechanisms responsible for enhancement by ATP remain to be clarified.
Implications of the H+ Current in BMMC Functions
Measurements of pHi using BCECF provide evidence
that the H+ conductive pathway is indeed responsible
for the rapid pHi recovery from acid-load in BMMC, although other unidentified mechanisms may be involved in the slowly developed pHi recovery. The current amplitude (9.3 ± 9.8 pA/pF at +100 mV and 32°C
with pHp/pHo of 5.5/7.3) of BMMC is smaller than
that in some phagocytes but is roughly comparable to
that reported in various cell types (DeCoursey and
Cherny, 1994). It is conceivable that metabolic acidosis
accompanies cell growth of BMMC as reported in other
cell types (Gerson and Kiefer, 1982
; Grinstein and
Dixon, 1989
). Otherwise, maintenance of pHi may be
challenged by releasing large quantities of chemical mediators during anaphylactic actions or in bacterial
infection (Echtenacher et al., 1996
; Malaviya et al.,
1996
). The H+ efflux mechanism dissipates metabolic
acids generated during these cellular activities (Lukacs
et al., 1993
). In addition, many mast cell activities are
pH dependent: for example, exocytosis is influenced by
pHi (Alfonso et al., 1994
), and enzymes, such as chimases, are strongly pH dependent (McEuen et al.,
1995
). Thus the H+ conductance may play a significant
role in pHi regulation of mast cells in various functional states, as in other cells (Hoffman and Simonsen,
1989).
Electrophysiological studies are often conducted at room temperature, which may cause one to overlook the H+ current. Our results imply that H+ conductive pathways are expected to exist more widely.
Original version received 31 October 1996 and accepted version received 18 March 1997.
Address correspondence to Miyuki Kuno, MD, Ph.D., Department of Physiology, Osaka City University Medical School, Abeno-ku, Osaka 545, Japan. Fax: 06-645-2015; E-mail: kunomyk{at}msic.med.osaka-cu.ac.jp
1 Abbreviations used in this paper: BCECF, 2We thank Dr. Matsuura for critically reading the manuscript.
This work was supported by the grants from The Ministry of Education, Science, Culture, Japan and from The Naito Foundation, The Ichiro Kanehara Foundation and The Hoansha Foundation.