(Received for publication, July 17, 1996, and in revised form, October 21, 1996)
From the Physiological Laboratory, Downing Street, Cambridge CB2 3EG, United Kingdom, and the § Department of Biochemistry, University of Glasgow, Glasgow G128QQ, United Kingdom
Previous reports have suggested that receptors
for immunoglobulin G (IgG), FcRs, directly activate a nonselective
cation channel (Young, J. D.-E., Unkeless, J. C., Young, T. M.,
Mauro, A., and Cohn, Z. A. (1983) Nature 306, 186-189;
Nelson, D. J., Jacobs, E. R., Tang, J. M., Zeller, J. M., and Bone,
R. C. (1985) J. Clin. Invest. 76, 500-507). To
investigate the mechanisms underlying membrane conductance changes
following human high affinity (Fc
RI) receptor activation, we have
used the human monocytic cell line U937 and combined conventional whole
cell patch-clamp recordings with single cell fura-2 Ca2+
measurements. Using a K+-free internal solution, antibody
cross-linking of IgG-occupied Fc
RI activated an inward current at
negative potentials, whose amplitude and time course mirrored the
concomitant rise in intracellular Ca2+. Current-voltage
relationships, obtained under different ionic conditions, revealed a
monovalent cation-selective conductance that, under physiological
conditions, would result in Na+ influx. Noise analysis of
current recordings indicated a single channel conductance of 18 picosiemens and a mean opening time of 4.5 ms. This current was also
activated by rises in intracellular Ca2+ induced by
ionomycin (3 µM) or thapsigargin (1 µM).
Addition of the Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N
,N
-tetraacetic acid to the intracellular medium abolished any channel activation by
ionomycin, Fc
RI, or the low affinity receptor, Fc
RII. These results demonstrate that Fc
RI activation triggers a novel
Ca2+-activated channel selective for monovalent cations and
that neither Fc
RI nor Fc
RII can directly activate a channel.
Human receptors for the constant, or Fc, region of immunoglobulin
G (IgG),1 FcRs, play a central role in
linking the cellular and humoral arms of the immune system and trigger
a number of downstream events including endocytosis, phagocytosis,
superoxide generation, and cytokine release (for reviews, see Refs.
1-3). Three closely related classes of Fc
Rs have been identified: a
high affinity (Fc
RI) and two low affinity (Fc
RII and Fc
RIII)
forms, each of which has different tissue distribution, structure, and
affinity for IgG (1). Both Fc
RI and Fc
RII are constitutively
expressed on cells of monocyte/macrophage lineage including the human
monocytic cell line U937, which has commonly been used as a model in
which to study Fc receptor signaling (4). Fc
RI is a 72-kDa protein comprising three extracellular immunoglobulin (Ig)-like domains of the
C2 set, a single transmembrane-spanning region, and a short cytoplasmic
tail with no known signaling motifs (5), whereas the 40-kDa class II
receptor contains only two Ig-like extracellular domains and has a
cytoplasmic region containing tyrosine kinase activation motifs (6).
Signaling by both receptors is thought to involve mainly, although not
exclusively, aggregation of tyrosine kinase activation motifs leading
to recruitment and activation of a number of soluble tyrosine kinases
and the subsequent initiation of various signaling pathways, for
example phospholipase C
activation (7).
Several reports suggest that FcRs could directly couple to and
activate a nonselective cation channel. Indirect measurements of mouse
macrophage membrane potential using
[3H]tetraphenylphosphonium ion accumulation indicated
that Fc
R activation triggered an initial
Na+-dependent depolarization followed by a
prolonged hyperpolarization in part attributable to K+
efflux (8). Subsequent recordings from planar lipid bilayers, into
which Fc
R-containing proteoliposomes (again from mouse macrophages) had been incorporated (9), suggested that the receptor was tightly
associated to and directly activating a nonselective cation channel.
More recently, in studies using human macrophages, antibody cross-linking of Fc
Rs (10) and Fc receptor-mediated phagocytosis of
opsonized particles (11) have been shown to trigger channel activation
and inward currents. In this study we have combined conventional whole
cell patch-clamp recording and single cell fura-2 Ca2+
measurements of U937 cells to examine the conductance changes following
Fc receptor activation and to determine the mechanism of channel
activation. Furthermore, we have investigated the possible functional
consequences of channel activation using measurements of membrane
potential and intracellular [Na+]. Preliminary results
from this study have appeared in abstract form (12).
U937 cells were cultured in a
humidified atmosphere at 37 °C, 6.8% CO2 in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal calf
serum, 2 mM glutamine, 10 units/ml penicillin, and 10 mg/ml
streptomycin. Cells were harvested by centrifugation, washed, and
resuspended in a standard external saline (see below). Ionomycin was
obtained from Calbiochem; fura-2, SBFI-AM, Pluronic F-127 (all prepared
as stocks in dimethyl sulfoxide), and Cs4BAPTA were from
Molecular Probes, Inc. Monomeric polyclonal human IgG and FcRII
monoclonal antibody were from Serotec (U. K.), and all other reagents
were from Sigma (U. K.). All experiments were carried out at room
temperature (20-23 °C).
Whole cell patch-clamp experiments were
carried out using an Axopatch 200A patch-clamp amplifier (Axon
Instruments, Foster City, CA). Pipettes were pulled from borosilicate
glass tubing (Clark Electromedical Instruments) and had filled
resistances of 2-3 megohms. Electronic compensation of capacitance
currents and series resistances (which were between 10 and 30 megohms) was performed. Membrane currents during voltage ramps were filtered at
2 kHz and sampled at 10 kHz using Axon Instruments hardware and pCLAMP6
software (Axon Instruments). Currents were also acquired continuously
at 37 kHz (filtered at 5 kHz) by a VR-10B digital data recorder
(Instrutech Corp.). Liquid junction potentials were measured by
reference to a 3 M KCl bridge and correcting computations made. Cells were resuspended in standard external saline containing (in
mM): 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose (pH 7.35, Tris). The internal
solution contained (in mM): 150 KCl, 1 MgCl2,
10 HEPES (pH 7.35, Tris). To minimize the contribution of
K+ currents, K+ was replaced internally by
Cs+, externally by Na+, and 10 mM
tetraethylammonium chloride was added externally. For low
Cl solutions, all Cl
, except that added
with divalent salts, was replaced by either aspartate or gluconate. For
solutions containing no monovalent cations, these were substituted by
NMDG+. High Ca2+ external solution contained
(in mM): 110 CaCl2, 10 HEPES (pH 7.35). Highly
buffered internal solution contained (in mM): 80 cesium
gluconate, 20 Cs4BAPTA, 5 NaCl, 0.2 Na2GTP. For
simultaneous fura-2 fluorescence experiments, 0.1 K5 fura-2
was added to the patch solution. In nystatin-perforated patch
experiments, the pipette contained (in mM): 100 KCl, 40 K2SO4, 1 MgCl2, 10 HEPES (pH 7.35, Tris). Cross-linking antibodies and ionomycin were applied from a
nearby pipette (150 µm from the cell) using a pressure injection
system (PLI-100, Medical Systems).
To activate FcRI, cells were
first loaded, for 15 min, with polyclonal human IgG (10 µM). The addition of goat anti-human IgG antibody (0.2 mg/ml) was then used to cross-link and thus activate IgG-loaded
Fc
RI. Since Fc
RI alone can bind monomeric IgG with significant
affinity, this established cross-linking method only results in Fc
RI
activation (for review, see Ref. 13). To activate Fc
RII
specifically, cells were preloaded with the mouse IgG1 anti-Fc
RII
monoclonal antibody, AT10 (10 µM) followed by subsequent
addition of goat anti-mouse IgG1 antibody (0.2 mg/ml).
Whole cell currents used for both
nonstationary fluctuation analysis and spectral analysis were obtained
at a holding potential of 28 mV with 140 mM sodium
aspartate, K+-free external solution, and cesium aspartate
internally. Recordings were filtered at 1 kHz (through an eight-pole
Bessel filter) and acquired at 5 kHz. For fluctuation analysis, the
mean current and its variance were calculated for 200-ms segments taken
once or twice every second before, during, and after the addition of cross-linking antibody. Spectral analysis, using Origin (Microcal, MA)
software, involved averaging the fast Fourier transform of 10 4,096-point segments taken during channel activation and subtracting the average fast Fourier transform of 10 taken before the addition of
cross-linker. The subtracted power spectrum was fitted by a single
Lorentzian function,
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
Single cell fura-2 fluorescence measurements were made using a Cairn Spectrophotometer system. Excitation light passed through a spinning filter wheel assembly containing four 340 nm and two 380 nm bandpass excitation filters. Emitted light was selected by two (400-600 nm) dichroic filters and further filtered by a 485 nm long pass gelatin filter and a 600 nm dichroic mirror. The combined output from all 340 and 380 nm excitation filters provided a 340/380 nm ratio for each revolution of the filter wheel. The signal was then averaged to give a ratio value every 67 ms. Background and cell autofluorescence were subtracted from the signal to give fura-2 fluorescence. [Ca2+]i was calculated according to Grynkiewicz et al. (14) using a Kd for fura-2 of 135 nM.
SBFI Fluorescence ExperimentsIn SBFI fluorescence experiments, cells were loaded by 45-min incubation at room temperature with 10 µM SBFI-AM mixed with an equal volume of 25% (w/v) Pluronic F-127. Aliquots of 106 cells were resuspended in 1.5 mM external saline (containing, in mM: 145 NaCl, 5 KCl, 1 MgSO4, 2 CaCl2, 10 glucose, 10 HEPES (pH 7.35, Tris) and 2 mg/ml bovine serum albumin), placed in continuously stirred cuvettes attached to a Cairn Spectrophotometer system (as above). Signal calibration was achieved by resuspending aliquots in media containing different [Na+] prepared by appropriate mixing of high [Na+] solution (110 mM sodium gluconate, 30 mM NaCl, 2 mM CaCl2, 10 mM Na-HEPES (pH 7.4)) with a similar solution in which K+ completely replaced Na+ (15). [Na+]i was clamped by the addition of a mixture of ionophores (5 µM each, gramicidin, nigericin, and monensin (15)), and the fluorescence ratios were calibrated as described by Harootunian et al. (16).
Under
pseudophysiological ionic conditions of 145 mM NaCl
externally and 150 mM KCl pipette solution, the addition of
goat anti-human antibody to IgG-loaded cells held at 20 mV resulted in the generation of a large transient outward current (current density
of 23.7 ± 5.6 pA/picofarad; n = 3). Voltage ramps
from
80 to +60 mV applied every 9 s during the experiment (shown
plotted against time in Fig. 1A) revealed the
development of a current, entirely outward over this voltage range,
which displayed a curvilinear current-voltage relationship. The current
reached a peak approximately 60 s after the addition of
cross-linking antibody and returned to resting levels after 2-3 min.
Since the size and duration of this current mirrored the
Fc
RI-induced rise in [Ca2+]i,
shown in Fig. 1B and reported previously in U937 cells (17),
and this current could be abolished by substitution of internal
K+ by Cs+ (data not shown), we concluded that
it was due to the activation of calcium-activated K+
channels known to be present in many monocytic cell types (18).
Under conditions that minimized the contribution of both K+
and Cl to whole cell current recordings (145 mM sodium aspartate, 10 mM tetraethylammonium
chloride externally, and 150 mM cesium aspartate internally; see "Experimental Procedures"), the addition of goat anti-human IgG antibody generated a small inward current in 23/30 cells
held at
40 mV (current density 1.148 ± 0.52 pA/picofarad, n = 12), which mirrored the concomitant rise in
[Ca2+]i (Fig. 1B). No
current activation or Ca2+ rise was observed in cells not
preloaded with polyclonal human IgG (n = 2, data not
shown). Leak-subtracted current-voltage relationships obtained during
and after current activation are shown in Fig. 1C and
demonstrate the development of a linear ("ohmic") conductance with
an Erev (where Erev = a reversal potential), under
these ionic conditions, of
16 mV.
To
determine whether this current was activated directly by FcRI or
required an increase in [Ca2+]i,
20 mM Cs4BAPTA was added to the pipette
solution to buffer any [Ca2+]i
rise. Under these conditions, the addition of goat anti-human antibody
both failed to trigger a [Ca2+]i
rise (Fig. 2A) and also prevented the
activation of any whole cell current (n = 11), as shown
by the leak-subtracted current-voltage curves (Fig. 2B).
Under identical conditions, specifically cross-linking Fc
RII using
monoclonal antibody (see "Experimental Procedures"), again failed
to activate a current (n = 9). In separate fluorescence
experiments, specific Fc
RII cross-linking was shown to trigger a
[Ca2+]i rise in intact cells (data
not shown). Leak-subtracted current-voltage relationships obtained 60 and 180 s following Fc
RII cross-linking are shown in Fig.
2C. These results indicate that neither Fc
RI nor Fc
RII
can directly activate a nonselective current in U937 cells.
To confirm that a rise in [Ca2+]i
was necessary and sufficient to activate a nonselective current, we
examined the effect of a 10-s application of the Ca2+
ionophore, ionomycin (3 µM), to whole cell current
recordings with Na+ aspartate externally and
Cs+ aspartate internally. In 6/6 cells held at 40 mV,
ionomycin activated an inward current (current density 2.53 ± 1.375 pA/picofarad). The leak-subtracted current-voltage curves,
generated by voltage ramps applied every 5 s following the
addition of ionomycin, are shown in Fig. 2D and demonstrate
the development of a nonselective conductance that, at high levels of
current activation, displays some inward rectification. Since these
recordings were obtained in the presence of 5 mM KCl, one
possibility was that this observed rectification was due to a small
K+ influx through Ca2+-activated K+
channels which, despite the presence of internal Cs+
(blocking K+ efflux), would still be predicted under these
conditions. This conclusion was supported by a similar experiment,
carried out in the absence of any external K+ (Fig.
2E), where the activated current showed a linear (ohmic) current-voltage relationship in the voltage range
80 to +60 mV.
An indication of the affinity of Ca2+ binding and the level of cooperativity involved in activation of this current was obtained by plotting the degree of current activation (under ionic conditions identical to those for Fig. 2E) as a percentage of maximum against [Ca2+]i, as determined by simultaneous fura-2 fluorescence measurements. The resultant plot (Fig. 2F) was fitted by a modified Hill equation
![]() |
(Eq. 3) |
The ion selectivity of this
Ca2+-activated conductance was investigated in a series of
ionic substitution experiments (Fig. 3 A-C).
Voltage ramps, applied throughout the experiments, were used to
generate leak-subtracted current-voltage relationships of the
FcRI-induced current as described above. When external monovalent
cations were substituted with the impermeant ion, NMDG+, an
outward current developed in response to a 10-s addition of either
cross-linking antibody (current n = 8; Fig.
3A) or 3 µM ionomycin (n = 12, data not shown). Substitution of both internal and external monovalent
cations with NMDG+ in the presence of 5 mM
Ca2+ externally resulted in no detectable conductance
change (n = 6; Fig. 3B), suggesting a
conductance permeable to monovalent cations and with little or no
permeability to Ca2+. To define the level of permeability
to divalents, 110 mM CaCl2 was used externally
with NMDG+ aspartate internally. The addition of 3 µM ionomycin (n = 5; Fig. 3C)
or cross-linking antibody (n = 3; data not shown)
failed to generate any detectable inward current. Ionomycin also failed to activate a current when the external solution was changed to 110 mM BaCl2 (n = 4, data not
shown), further indicating no significant permeability to divalent
cations.
Single Channel Properties
The mean single channel conductance
of this current was estimated by nonlinear fluctuation analysis. The
variance and mean current were calculated for 200-ms segments taken
once every 0.5 or 1 s during low levels of channel activation
(Fig. 4A). The variance is shown in Fig.
4A, and the variance plotted against the mean current in
Fig. 4B. Although the relationship of current variance with
mean current over the entire range of opening probabilities is best
described by a binomial distribution (19), at low levels of channel
activity (i.e. when channel opening follows a Poisson distribution), it is approximately linear with a slope equal to the
mean single channel current (19, 20). A straight line, fitted to the
data in Fig. 4C by linear regression), gave a single channel
current of 218 fA. With a holding potential of 28 mV and a reversal
potential under these ionic conditions, of
16 mV, this corresponded
to a unitary conductance of 18 pS (n = 2). This may be
an underestimate since this method is known to generate lower values
than direct single channel recording (21).
We went on to use spectral analysis to determine the mean channel opening time. The background corrected spectrum (Fig. 4C) was well fitted by a single Lorentzian function with a corner frequency of 35 Hz. This corresponded to a single open state with a mean opening time of 4.5 ms. The total variance calculated from this background-corrected spectrum (1.37 pA2) agreed well with the variance of the mean current, obtained by fluctuation analysis, in the same experiment (1.1 pA2), confirming that the power spectrum obtained was dominated by noise attributable to the nonselective cation channel.
FcOne expected consequence of activation of this
monovalent-selective channel under physiological conditions will be to
cause an influx of Na+. We examined the magnitude of
changes in [Na+]i using population
fluorescence recordings of cells loaded with the Na+
indicator, SBFI (Fig. 5A). Cross-linking
FcRI triggered a slow rise in
[Na+]i reaching a peak of
18.7 ± 3.1 mM (n = 3) after
approximately 4 min and returning to basal levels after 20-25 min. To
assess whether this Na+ influx would result in a membrane
depolarization, current-clamp recordings were made under nystatin whole
cell configuration. Fig. 5B shows a typical experiment.
Resting membrane potentials of
21.0 ± 8.29 mV
(n = 7) were recorded. These values compare with
previous measurements in monocytes and macrophages of between
15 and
56 mV (10; for review, see Ref. 18). Following a short delay, the
addition of cross-linking antibody resulted in membrane hyperpolarization to
64.7 ± 6.07 mV (n = 7)
lasting 2-3 min.
The present study demonstrates that FcRI cross-linking triggers
a Ca2+-activated cation channel, highly selective for
monovalent over divalent ions, with a unitary conductance of 18 pS. In
addition, we have shown that neither the high affinity (Fc
RI) nor
low affinity (Fc
RII) forms of the IgG receptor can directly activate
a nonselective cation channel in U937 cells. There have been numerous
reports of various types of Ca2+-activated nonselective
cation (CAN) channels in a wide variety of cells types (for review, see
Ref. 22). These channels show considerable variation in unitary
conductance (18-45 pS), mean opening time (0.5-930 ms), and
Ca2+ sensitivity for activation (50 nM-1
mM). In addition, there seems to be a division between CAN
channels showing some permeability to divalent as well as monovalent
cations and those that show no detectable permeability to
Ca2+ (22). Thus the Ca2+-activated channel
found in U937 cells appears to belong to this last group. A
Ca2+-activated channel with similar selectivity for
monovalents and single channel conductance (22 pS) has been reported in
neuroblastoma cells (23); however, the Ca2+ sensitivity for
activation (Kd of 1 µM) and mean
single channel opening time (50-200 ms) vary considerably from those observed for the channel in U937 cells (Kd for
Ca2+ activation: 278 nM; mean opening time: 4.5 ms).
The properties of the Ca2+-activated monovalent cation
channel in U937 cells are similar to those of Fc receptor-operated
cation channels reported by other groups. The direct Fc
receptor-operated channel reported from lipid bilayer studies (9) has
similar ionic selectivity (low Ca2+ permeability) and a
slightly larger single channel conductance (50 pS). In single cell
studies where Fc receptor cross-linking, by antibody (10) or opsonized
particle (11), has been shown to trigger a current attributable to
Na+ influx, intracellular Ca2+ changes were not
monitored or prevented. This raises the possibility that channel
activation was via a rise in
[Ca2+]i and not directly
receptor-triggered. Inside-out patch recordings from mouse macrophages,
excised after FcR-evoked channel activity had been observed in a
cell-attached configuration (24), showed the presence of a nonselective
cation channel with a 35-45-pS single channel conductance whose
opening could be modulated by [Ca2+]i. Neither Ca2+
activation in the absence of receptor cross-linking nor
Ca2+ permeability was assessed; however, it seems likely
that this channel is similar to the one reported in this study.
Whole cell current recordings indicated that activation of
Ca2+-dependent K+ channels was the
dominant ionic conductance change following FcRI stimulation of U937
cells under pseudophysiological conditions (see Fig. 1A).
This accounts for the prolonged hyperpolarization of more than 45 mV
observed in current-clamp recordings of membrane potential (Fig.
5B). The lack of observable depolarization can be explained
by both the opposing action of K+ current through
Ca2+-activated K+ channels and the fact that
the resting potential (
21 mV) is very close to the reversal potential
for this Ca2+-activated cation channel (
16 mV).
Differences in resting potentials and variations in the relative
density and/or differences in Ca2+-binding affinities of
CAN and Ca2+-activated K+ channels may explain
the initial depolarization and subsequent hyperpolarization observed in
mouse macrophages (8) and the transient outward current followed by a
sustained inward current reported in human alveolar macrophages (10)
following Fc
R activation. Indeed, earlier microelectrode studies
reporting action potentials in human monocyte-derived macrophages (25)
may also be explained along similar lines.
The possible physiological role for this CAN channel remains unclear.
One predicted consequence of channel activation would be to cause a
Na+ influx that would be enhanced under conditions of
membrane hyperpolarization. Fluorescence measurements of SBFI-loaded
U937 cells (Fig. 5A) revealed an FcR-triggered
[Na+]i rise of 10-20
mM. The contribution of CAN channel activation to this
[Na+]i can be estimated by
integration of Fc
RI-evoked currents generated under the ionic
conditions in Fig. 1B and scaled to holding potentials of
75 mV. This is the potential observed in current-clamp experiments
following cross-linking antibody addition (Fig. 5B). Using
this method, the total Na+ influx through CAN channels
following Fc
RI activation was obtained and provided an estimated
[Na+]i increase, for an 8-µm
cell, of 31.6 ± 3.3 mM. This value is considerably
greater than peak Na+ concentrations obtained by SBFI
fluorescence measurements (18.7 mM) and indicates that the
CAN channel can account for most of the observed Na+
influx. However, it also suggests that some Na+ efflux must
take place. Further studies will be required to assess the
contributions of other influx pathways, such as
Na+/Ca2+ or Na+/H+
exchangers, to this [Na+]i rise.
Increases in [Na+]i have been
reported to alter cytosolic pH (26) and osmolarity (27) required for
cytoskeletal rearrangement in a variety of cell types, modulate G
protein receptor coupling (28), and alter K+ channel
activity (29). This raises the possibility that one or more downstream
events initiated by Fc
R activation may require or be modulated by
rises in [Na+]i.
In conclusion, we have shown that in U937 cells, Fc receptor
aggregation does not activate a conductance directly but triggers a
Ca2+-activated cation channel, selective for monovalents,
which contributes to FcR-mediated Na+ influx.
We thank Dr. S. O.Sage for helpful comments.