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Vol. 55, Issue 6, 970-981, June 1999
3/
4 Neuronal Nicotinic Acetylcholine Receptor
Department of Pharmacology, Georgetown University School of Medicine, Washington, D.C.
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Summary |
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The
3/
4 rat neuronal nicotinic acetylcholine receptor, stably
transfected in human embryonic kidney cells, was examined using
the whole-cell-clamp technique and 2-dimensional confocal imaging. Application of agonists (nicotine, cytisine, epibatidine) activated a large (100-200 pA/pF) inwardly rectifying monovalent current, with little current at voltages between 0 and +40 mV. Rapid
application of nicotine and cytisine indicated EC50 values of
22 and
64 µM, respectively, and suggested second order
binding kinetics (Hill coefficient ~2). The time constant of
desensitization (decay) of nicotine-activated current was
concentration-dependent (typically ~10 s at 30 µM versus ~1.0 s
at 100-1000 µM), but not voltage-dependent and was significantly
smaller than the ~200 s reported for the
3/
4 receptor expressed
in Xenopus oocytes. Nicotine-activated current was
rapidly and reversibly blocked by coapplication of mecamylamine and
d-tubocurarine. At
80 mV holding potentials, the
current was also suppressed by ~25% either upon complete removal or
elevation of Ca2+ to 10 mM. Total replacement of
Na+ by Ca2+ also completely blocked the
current. On the other hand, evidence for permeation of Ca2+
was indicated by increased inward current at
40 mV upon elevation of
Ca2+ from 2 to 10 mM, as well as a rise in the cytosolic
Ca2+ proportional to the current carried by the receptor.
These findings are consistent with the idea that Ca2+, in
addition to its channel-permeating properties, may also regulate the
receptor from an extracellular site. Our results suggest that the
3/
4 neuronal nicotinic acetylcholine receptor, when stably expressed in human embryonic kidney 293 cells, has desensitization kinetics and Ca2+ regulatory mechanisms somewhat different
from those described for the receptor expressed in
Xenopus oocytes.
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Introduction |
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Neuronal
nicotinic acetylcholine receptors (nAChRs) composed of
and
subunits are differentially expressed in the nervous system and
constitute a family of ligand-gated cationic channels. Eight
(
2-
9) and three
(
2-
4) neuronal subunit genes have been cloned in chicks, rodents, and humans, consistent with the idea
that neurons may express nAChR with a variety of subunit combinations
(see McGehee and Role, 1995
; Lindstrom, 1996
, for recent reviews).
The central nervous system expresses mRNA for all of the nAChR
subunits. The receptors that contain the
3 subunit, although lower
in abundance than
4, may mediate important effects of acetylcholine and nicotine in the brain (Connolly et al., 1995
) and possibly in the
spinal cord. Compared with
4/
2 receptors, nAChR containing
3
subunits appear to have much lower affinity for most nicotinic agonists
in the central nervous system (Albuquerque et al., 1997
).
The nAChR comprised of
3 subunits in combination with either
2 or
4 subunits may be the predominant nicotinic receptor in the
mammalian sympathetic, parasympathetic, and trigeminal ganglia, and in
adrenal chromaffin cells (Nooney and Feltz, 1995
). Rat and chicken
superior cervical ganglion, chick ciliary ganglion, and PC12 cells also
express mRNA for designated
3,
5,
2, and
4 subunits
(McGehee and Role, 1995
).
Expression studies in Xenopus oocytes have shown that
functional neuronal nAChRs can be formed by pairwise coinjection of one
kind of
and one kind of
subunit (Boulter et al., 1987
). The
receptor forms with an apparent subunit stoichiometry of two
and
three
(Anand et al., 1991
) or, in some cases (
7 and
8), as
homomeric assemblies (Séguéla et al., 1993
). Both
and
subunits contribute to the pharmacological and biophysical
properties of these receptors, giving each subunit combination unique
characteristics (Wada et al., 1988
; Luetje and Patrick, 1991
).
In this report we explore the biophysical and pharmacological
characteristics of the
3/
4 nAChR subtype in a stably transfected mammalian human embryonic kidney (HEK) 293 cell line recently developed
in our laboratory (Xiao et al., 1998
). Our results suggest that
3/
4-transfected cells generate a rapidly activating,
voltage-dependent monovalent current on exposure to nicotinic receptor
agonists. The current shows characteristic inward rectification,
desensitization, kinetics, and pharmacology similar but not identical
with the neuronal nAChR in chromaffin cells. The electrophysiological
and confocal Ca2+ imaging data suggest that
Ca2+ may both permeate and allosterically
regulate the
3/
4 receptor (Lena and Changeux, 1993
).
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Experimental Procedures |
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Materials.
Stably transfected HEK 293 (ATCC CRL
1573)-expressing
3/
4 neuronal nAChRs (cell line designation:
KX
3
4R2 cells) were prepared as described
previously (Xiao et al., 1998
). Cells were plated in tissue culture
medium (Gibco-BRL, Gaithersburg, MD) containing bovine serum and
antibiotics. ACh-, nicotine, and
d-tubocuraine (d-tc) were obtained from Sigma Chemical Company (St. Louis, MO). Mecamylamine and epibatidine were
purchased from Research Biochemicals (Natick, MA). A similar stable
transfection of the rat
3/
4 receptor in HEK 293 cell line has
been reported previously (Stetzer et al., 1996
).
Cell Transfection and Culture.
HEK 293 cells were maintained
at 37°C with 5% CO2 in the incubator. Growth
medium for HEK 293 cells was minimum essential medium supplemented with
10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin.
Stably transfected cell lines were raised in selective growth medium
containing 0.7 mg/ml of Geneticin (G418). Transfection was conducted by
the calcium phosphate method (Chen and Okayama, 1987
). Briefly,
exponentially growing HEK 293 cells were plated in 100-mm dishes
containing 10 ml of the growth medium 24 h before transfection.
For transfection 1.0 ml of solution containing 10 µg of linearized
DNA, 125 mM CaCl2, 25 mM HEPES, 140 mM KCl, 6 mM
glucose, 0.75 mM Na2HPO4
(pH 7.05) was added to the cell-containing dish in drop-wise fashion.
The cells were incubated with the transfection mixture for 16 h in
the incubator and then were grown in fresh growth medium for 24 h.
They were collected and plated at a range of densities. The selection
process was continued for 3 to 4 weeks. G418-resistant clones were
picked up by cloning cylinders and have been maintained in continuous culture for about 1 year.
Electrophysiological Recording.
Functional expression of
nicotinic receptors was evaluated in the whole-cell configuration of
the patch clamp technique using a DAGAN 8900 amplifier (Dagan Corp.,
Minneapolis, MN). The patch electrodes, pulled from borosilicate glass
capillaries, had a resistance of 3 to 5 M
when filled with the
internal solution containing (in mM): CsCl, 110; tetraethylammonium
chloride, 20; NaCl, 0 to 20; MgATP, 5; EGTA, 14; HEPES, 20 and titrated
to pH 7.2 with CsOH. About 90% of the electrode resistance was
compensated electronically, so that effective series resistance in the
cell-attached configuration was always less than 1 M
. Stably
transfected HEK cells were studied 2 to 4 days after plating the cells
on coverslips. Generation of voltage-clamp protocols and acquisition of
data were carried out using pCLAMP software (Axon Instruments, Inc., Burlingame, CA). Sampling frequency was 0.5 to 2.0 kHz and current signals were filtered at 10 kHz before digitization and storage. All
experiments were performed at room temperature (23-25°C). Currents
were normalized relative to the membrane capacitance (10-30 pF) and
then calculated as the mean ± S.E.M. for the number of cells
examined (n).
Perfusion System.
Cells plated on plastic coverslips (15 mm
round thermanox, Nunc, Inc., Napierville, IL) were transferred to an
experimental chamber mounted on the stage of an inverted microscope
(Diaphot, Nikon, Nagano, Japan) and were bathed in a solution
containing (in mM): NaCl, 137; CaCl2, 2; HEPES,
10; glucose, 10; MgCl2, 1 (pH 7.4 with NaOH). The
experimental chamber was constantly perfused at a rate of about 1 ml/min with the control bathing solution. The amplitude and time course
of the nicotinic current was highly dependent on the speed of
application of nicotine. A reduction in flow rate significantly slowed
the activation, decreased the amplitude, and slowed the desensitization
of the nicotinic current (Callewaert et al., 1991
). We therefore used
servo-controlled miniature solenoid valves for rapid switching between
control and test solutions (Cleemann and Morad, 1991
). The effective
switching time was determined in rat ventricular cells by measuring the peak Na+ currents at various times after
triggering a change in
[Na+]o or by measuring
the holding current at the tip of an open patch pipette subjected to a
solution of low Cl- (Davies et al., 1988
). Under
optimal conditions, such changes in solution had a delay of 6 to 8 ms
(corresponding mainly to the pull and release of the solenoid valves
and replacement of fluid in the common outlet of the perfusion
manifold) followed by a transition period where the measured current
changed with a time constant of 5 to 10 ms. In repeated applications
the delay was fairly reproducible, but it did show some variation in
different experiments with different hydrostatic pressures and
perfusion manifolds. The transition time was typically about 20 ms.
Confocal Microscopy.
Nicotine-induced
Ca2+ signals in single voltage-clamped HEK cells
were measured by adding 1 mM of the fluorescent
Ca2+ indicator dye fluo-3 to the dialyzing
pipette solution and scanning the cell at a rate of 30 frames per
second with a confocal microscope (Odyssey XL, Noran Inc., Madison, WI;
microscope: Zeiss, Axiovert 135, 40× Zeiss 440052 c-apochromat
objective, NA. 1.2). At this rate, the microscope collects full
two-dimensional frames of 640 × 480 pixels on a 0.207 µm grid
by collecting data at 10 MHz and sweeping the rapidly scanned direction
with an acousto-optical deflector at 17 kHz. Detected fluorescent light
passes through a variable slit extending in the rapidly scanned
direction so that the instrument is confocal only in the slowly scanned
direction. Ca2+ signals were measured as the
change in fluorescence intensity over the entire cell, or they were
normalized relative to the fluorescence intensity before application of
nicotine (
F/F). Cells examined with confocal microscopy were
cultured on 25-mm glass coverslips and subjected to reduced flow rates.
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Results |
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Agonist-Induced Current.
Stably transfected HEK 293 cells
expressing
3/
4 nAChR generated rapidly activating inward current
at
60 mV holding potentials when exposed to small concentrations of
nACh receptor agonists. Figure 1 compares
representative traces of current activated by application of 10 µM
ACh (A), 10 µM nicotine (B), and 10 nM epibatidine (C). One
micromolar concentrations of atropine were used to block any
endogenous muscarinic receptors when ACh was used as an agonist. Because the time courses of activation and deactivation of the current
on application and withdrawal of nicotinic agonists (Fig. 1) are
significantly slower than the step change in agonist concentration (~20 ms; see Fig. 1B of Davies et al., 1988
; Tang et al., 1989
), it
is possible that the time courses of rise and fall of the current reflect the binding or gating kinetics of the nAChR channel. Comparison of activation and deactivation of the current shows clearly that epibatidine-evoked currents decayed significantly slower on removal of
the drug than those induced by acetylcholine, nicotine, and cytisine
(Figs. 1 and 2, A and B), consistent with
the higher binding affinity of the drug to the receptor (Xiao et al.,
1998
). Comparison of the peak currents generated by epibatidine and
nicotine at
60 mV holding potentials showed that 10 nM epibatidine
generated currents (88.3 ± 24.8 S.E.M. pA/pF, n = 5) equivalent to those activated by 200 µM ACh (93.0 ± 34.5 pA/pF, n = 4).
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Dose-Response Relation.
Figure 2 compares the dose dependence
and kinetics of activation of
3/
4 nAChR to rapid application of
various concentrations of nicotine and cytisine. The dose-response (D)
was measured by exposing each cell to at least three concentrations of
either nicotine or cytisine. Nicotine and cytisine at concentrations of
~1 µM failed to activate a detectable current in
3/
4-expressing cells. At higher concentrations (10-1000 µM)
nicotine and cytisine evoked a rapidly activating inward current that
varied in its magnitude and gating kinetics with concentration. The
peak amplitude of nicotine- and cytisine-activated currents at
80 mV,
when plotted as a function of agonist concentrations, showed that
nicotine and cytisine at concentrations of 100 to 200 µM generally
produced their maximal responses. The dose-response relationships were normalized and fitted with the empirical Hill equation
y = 1/(1+((EC50/[agonist])n))
yielding a cooperativity factor (n), which was close to 2 for both
agonists (nicotine: 1.96 ± 0.30; cytisine: 2.05 ± 0.17). The data indicate an EC50 of 22 ± 2 µM
for nicotine and 64 ± 7 µM for cytisine. The magnitude of the
fully activated current (corresponding to unity in Fig. 2D) was
121 ± 16 pA/pF (n = 14) in the group of cells
activated by nicotine and 182 ± 20 pA/pF (n = 11)
in the cells activated by cytisine, suggesting about equal efficacy for
the two compounds.
Voltage Dependence of Agonist-Induced Current.
Figure
3 compares the voltage dependence of
three nAChR agonists. Comparison of superimposed tracings of the
current activated with different agonist concentrations and test
potentials shows that: 1) agonist-activated currents decay little at
low concentrations and moderate holding potentials (~
40 mV) during
a 1.5-s drug exposure period; 2) at higher concentrations, the current
decays more rapidly after its activation; 3) little or no current is activated at 0 to +40 mV even when
[Na+]i was increased from
0 to 20 mM (data not shown); 4) the deactivation times increase at high
agonist concentrations; and 5) the voltage dependence of the
agonist-induced current displayed prominent inward rectification (Fig.
3, A-C, a characteristic of the whole-cell neuronal nAChR current
measured in sympathetic neurons, Mathie et al., 1990
; adrenal
chromaffin cells, Callewaert et al., 1991
; Nooney et al., 1992
; and
cultured hippocampal neurons, Bonfante-Cabarcas et al., 1996
; or in HEK
cells transiently transfected with
3/
4 nAChR, Wong et
al., 1995
).
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4/
2-expressing HEK cells (Buisson et al., 1996Decay of Agonist-Activated Current.
Nicotine-activated current
decayed significantly in the presence of the drug, resulting in part
from desensitization of the
3/
4 receptor. Time constants of the
decay of the current in the presence of nicotine typically ranged
between 1.0 and 10.0 s, depending on the agonist concentration
(Fig. 4A and B). After activation of the
receptor by 30 µM, 100 µM, and 1 mM nicotine, the time constant of
the decay of the current at
80 mV was found to be respectively
11 ± 6 (n = 3), 1.4 ± 0.3 (n = 5), and 2.0 ± 0.4 (n = 4) s
(
, Fig. 4B). The rate constant of decay of the current was mostly
independent of membrane potential (
,
120 mV;
,
80 mV;
,
40 mV), but increased with increasing concentration (Fig. 4B).
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60 mV, was
absent at voltages between
40 to +80 mV, was highly
concentration-dependent, and appeared to be somewhat enhanced at higher
concentrations of Ca2+ (Fig. 7A). A detailed
analysis of the rebound current in muscle end-plate acetylcholine
receptor has been recently reported by Maconochie and Steinbach (1998)
Fig. 4B). The cells on a given day of
experimentation would all have either slow or fast desensitization.
During an experiment lasting 5 to 20 min the time constant of
desensitization would often decrease by a factor of 2, but not enough
to alter a slowly desensitizing cell to a fast one.
Nicotine-Activated Current Was Blocked by Mecamylamine and
d-tc.
To further characterize the properties of the
nicotine-induced current in
3/
4-expressing cells, we examined the
kinetics, voltage, and concentration dependence of two well known nAChR blockers. To determine the relative speed of the antagonist action vis-à-vis the agonist-activated current, nicotine and the
antagonist were coapplied to the cell. Rapid application of
mecamylamine (1 µM) simultaneous with 10 µM nicotine suppressed the
nicotine-induced current by
80% (n = 4; Fig.
5A and C). The suppressive effect of the
drug was voltage-dependent such that at
120 mV, mecamylamine blocked the current by ~80% versus ~60% at
80 mV (Fig. 5B). Mecamylamine also significantly enhanced the decay kinetics
of the nicotine-activated current, consistent with the open
channel-blocking property of the drug (Fig. 5A). Nicotine-activated current recovered rapidly upon washout of the blocker. Thus,
mecamylamine appears to access the channel pore in a voltage- and
time-dependent manner. The kinetics of the block at the 1 µM
concentration were sufficiently fast as to prevent most of the
nicotine-activated current when the agonist and antagonist were
coapplied.
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Calcium Permeation and Block of Rat
3/
4 nAChR.
Reliable
measurements of reversal potential are required to assess directly the
ionic selectivity of agonist-induced current. Because little or no
outward current could be measured on activation of the nicotinic
receptor between 0 and +40 mV, the measurements of reversal potentials
were less reliable. Thus, we attempted to evaluate the permeation of
Ca2+ based on the larger current measured at very
negative and positive potentials.
3/
4 nAChR was modified when the external
Ca2+ concentrations were increased from 2.0 to 10 mM during application of 50 µM nicotine. At
120 mV and
80 mV the
higher [Ca2+]o suppressed the
current and the washout of the drug was accompanied by a noticeable
rebound current, as if the blocking effect of Ca2+ washed out before the deactivation of the
channel. In addition, the current activated by 50 µM nicotine, at
80 mV, was slightly suppressed when
[Ca2+]o was reduced to 0.2 mM (
,
Fig. 7B). This trend became significant in completely
Ca2+-free solution over a wide range of nicotine
concentrations (20 (
), 200 (
) µM nicotine). Significant
suppressions were also observed at 5 and 10 mM
[Ca2+]o. Complete
replacement of all the external NaCl with 90 mM
CaCl2, completely blocked the inward current
through the receptors during the drug exposure period (Fig. 7B) and
suppressed the outward current at +120 mV to 18 ± 11%
(n = 5) of values measured with 2 mM
[Ca2+]o. Thus, if
Ca2+ in addition to its blocking effect were also
to permeate the channel, it would most likely generate significant
current at the moderate Ca2+ concentrations.
Accordingly, at 10 mM Ca2+ (Fig. 7C), the
nicotine-induced currents appeared to decrease at
120,
80, and +120
mV, increase at
40 mV and remain undetectable at 0 and +40 mV. These
changes were significant when measured relative to the current recorded
in control solution with 2 mM Ca2+. The enhanced
inward current at
40 mV suggest that Ca2+ may
permeate through the nAChR channel and shift the reversal potential
toward more negative potentials.
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F/F) rises
continuously during the application of nicotine and decays slowly upon
removal of nicotine. Comparison of total charge carried by the
nicotinic channel in five different cells (represented by different
symbols in Fig. 8C) and change in fluorescence (
F/F) showed direct
correspondence of current and
F/F (Fig. 8C), suggesting that the
rise in Ca2+ is directly correlated to the ionic
charge transported by the
3/
4 receptor. Mecamylamine strongly
suppressed the nicotine-activated current and simultaneously inhibited
the rise in [Ca2+]i (Fig.
8C, open symbols), suggesting that the nicotinic channel was
responsible for the rise in Ca2+.
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80-mV holding
potentials, when K+ was substituted as
extracellular charge carrier for Na+. In a set of
three experiments, we found that nicotine-activated currents and the
rise in cytosolic Ca2+ were similar whether
Na+ (Control) or K+ (KCl)
was the charge carrier through the channel (Fig. 9). The inset frames
of Fig. 9 show that the ratiometric Ca2+ signals
(
F/F) increase in strength toward the distal ends of the cellular
protrusions more rapidly than the center of the cell and confirm that
K+ substitution alters neither the magnitude nor
the cellular distribution of the Ca2+ signals.
Because K+ is known not to substitute for
Na+ on the
Na+-Ca2+ exchanger, this
finding strongly suggests that the Ca2+ signals
are not produced secondary to influx of Na+, but
are directly related to the permeation of Ca2+
via the nAChR channel.
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Discussion |
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In response to application of nicotinic agonists (ACh, nicotine,
cytisine, and epibatidine), HEK cells stably transfected with rat
3/
4 nAChR generate a large rapidly activating and slowly decaying
current. This current displayed inwardly rectifying properties (generating little or no current between 0 and +40 mV, Fig. 3) and was
rapidly and reversibly blocked by mecamylamine and d-tc (Figs. 5 and 6). The nicotine-activated channel, at physiological Ca2+ concentrations, appears to transport
sufficient Ca2+ as to induce significant rise in
cytosolic Ca2+ concentrations (Figs. 8 and 9). At
higher concentrations, Ca2+ appears to suppress
the Na+ current through the receptor, consistent
with Ca2+ serving as a permanent blocker, but the
complete block of the channel on isoosmolar replacement of
Na+ by Ca2+ may suggest an
additional regulatory role for extracellular
Ca2+.
Dose-Response of Nicotine and Cytisine.
Nicotine and cytisine
appeared to activate the
3/
4 recombinant receptor with
EC50 values of about 22 and 64 µM,
respectively, and with a cooperativity factor of ~2.0 for both
agonists. Hill coefficients ranging between 1.5 and 2.0 have been
reported previously for both the recombinant and wild types of nAChR
(Lindstrom, 1995
; Wong et al., 1995
). Significantly lower Hill
coefficients of 0.6 and 1.4 were recently reported for the human
3/
4 recombinant receptor stably expressed in HEK cells
(Stauderman et al., 1998
). However, in those experiments, the
coefficients were derived from measurements of intracellular
Ca2+ in large populations of cells in response to
slow application of nicotinic receptor agonists and could reflect both
desensitization of the receptor and indirect functional coupling
between activation of nicotinic receptor and rise in
[Ca2+]i. Our
electrophysiological measurements, based on rapid application of
agonists, on the other hand, appear to fit a Hill coefficient close to
2.0 for both cytisine and nicotine (Fig. 2) as found also with human
3/
4 expressed in oocytes (Chavez-Noriega et al., 1997
). Cytisine
is generally reported to be at least as potent as nicotine in
activating the
3/
4 receptor to generate inward current in oocytes
(Chavez-Noriega et al., 1997
; Gerzanich et al., 1998
) or
Ca2+ influx in HEK cells (Stauderman et al.,
1998
). In our experiments, we found a somewhat higher
EC50 value for cytisine (Fig. 2D). However, this
may be in part due to measuring the current shortly after the drug
application and before the development of agonist-induced block or
desensitization (Fig. 2, A-C). Because such rapid measurements are not
possible in whole oocytes or dense populations of eukaryotic cells
where average activation times of the receptor are significantly slower
(>5 s), the previously reported dose-response relations may reflect
the steady-state component of the agonist-activated current.
Decay of Nicotine-Activated Current. The rate of decay of nicotine-induced current in the presence of nicotine was strongly concentration-dependent, such that at low concentrations of agonists (10-30 µM) time constants in excess of 10 s were required for full decay of the current, whereas at 100 to 1000 µM drug concentrations the current decayed within 1 to 2 s (Fig. 4).
Although it is tempting to attribute the decay of the current entirely to the desensitization of the receptor, the decay of current in the presence of the agonist may also reflect, in part, agonist-induced block of the channel. This might be particularly the case at higher concentrations of agonists, as demonstrated for the muscle endplate nicotinic receptors (Sine and Steinbach, 1984
3/
4-expressing HEK cells in our laboratory using
Rb+ efflux to measure channel function (Xiao et
al., 1998
3/
4-generated current (Fig. 3) and the
enhancement of "rebound" current at negative potentials (Figs. 3B,
4A, and 7A). Our data are, therefore, consistent with the idea that at higher concentrations and negative potentials ACh, nicotine, and cytisine may gain access to the channel permeation sites and serve as
open channel blockers (see scheme proposed by Colquhoun et al., 1987
3/
4-expressing HEK cells with those of primary cultures of adult
rat chromaffin cells exposed to the same set of experimental conditions
and techniques suggests significantly slower decay kinetics of ACh- or
nicotine-activated current in recombinant versus the native receptor.
The time constant of decay of nicotinic current in primary cultures of
rat chromaffin cells was 324 ± 58 ms (n = 5) when
activated with 30 µM and 128 ± 26 ms (n = 5) with 1 mM nicotine compared with 10.9 ± 5.9 s
(n = 3) and 2.1 ± 0.3 s (n = 4), respectively, in transfected HEK cells expressing the
3/
4
recombinant receptor.
In some populations of cells, the rate of decay of the current was much
faster, by as much as an order of magnitude (cf. Fig. 4, A and C),
approaching the decay kinetics of the current recorded in primary
cultures of rat chromaffin cells (Callewaert et al., 1991
3/
4 current reported here are
significantly faster than those recently reported for this receptor expressed in Xenopus oocytes. At 50 µM nicotine
concentrations, time constants of about 200 s were measured in
oocytes (Fenster et al., 1997Ca2+ Permeability of
3/
4 Receptor.
Permeation of Ca2+ through the native and the
recombinant nicotinic receptor has been the subject of considerable
interest and investigation. From the shift in the reversal potential,
on fractional increases of extracellular Ca2+, a
PCa/PNa of 0.9 for
3/
4 and 0.1 for the muscle
1/
1/
/
expressed in oocyte
have been estimated (Costa et al., 1994
). Direct comparison of charge
transported by the channel and the rise of intracellular
Ca2+ measured in chromaffin cells and in oocytes
expressing
3/
4 suggest that approximately 2 to 4% of the total
charge may be transported by Ca2+ at
physiological Ca2+ concentrations (Vernino et
al., 1992
; Decker and Dani, 1990
). Unexpectedly, however, Vernino et
al. (1992)
also found that a 10-fold increase in
[Ca2+]o also enhanced the
outward current through the
3/
4 receptor at positive (+30 mV)
membrane potentials, where enhancement of inward
Ca2+ current was expected (see also Amador and
Dani, 1995
). These findings support the idea that
Ca2+ may both permeate and allosterically
regulate the nAChR from an extracellular site (Lena and Changeux,
1993
). Consistent with a possible regulatory role of
Ca2+ on nAChR, Buisson et al. (1996)
reported a
50% suppression of the current on a 10-fold increase of
[Ca2+]o in
4/
2-transfected HEK cells. The decrease in the whole-cell current
appears to be carried by suppression of the single channel conductance
on elevation of [Ca2+]o
in both the native and recombinant receptors (Vernino et al., 1992
;
Mulle et al., 1992
; Amador and Dani, 1995
; Buisson et al., 1996
).
3/
4-transfected HEK cells showed
a bell shaped response for the magnitude of the current versus the
extracellular Ca2+ concentration (Fig. 7B). The
reduction of the current at low Ca2+
concentrations (Fig. 7B) may in part reflect a surface charge effect
that would shift the current-voltage relations to the left when the
divalent cation concentrations were lowered. The elevations of
Ca2+ from 2 to 10 to 90 mM partially or
completely (Fig. 7B) blocked the nicotine-activated current, consistent
with the data in
4/
2-expressing HEK cells (Bouisson et al.,
1996
40 mV. This finding is consistent with the
measurements in oocytes where the currents are large enough to reveal
the effects of Ca2+ at and near the reversal
potential (Gerzanich et al., 1998
120 mV and +120 mV.
Direct measurement of intracellular Ca2+ (Figs. 8
and 9) shows that the rise in intracellular Ca2+
was directly related to the magnitude of the charge transferred through
the receptor (Fig. 8, a 10-30% rise of
F/F for currents ranging
from 1.5-4 nA). Because these cells were dialyzed with 1 mM fluo-3
(Kd
300 nM) and assuming resting
Ca2+ activity of
100 nM, the predicted rise in
[Ca2+]i is about 33 to
100 µM, producing PCa/PNa
1, consistent with the
PCa/PNa values measured in
native cells or
3/
4 receptor expressed in oocytes (Costa et al.,
1994
80 mV and
when Na+ was replaced by K+
as the charge carrier (Fig. 9), however, suggests that
Ca2+ permeates directly through the nAChR
channel. Thus, fairly small and difficult to detect
Ca2+ currents through the
3/
4 receptor may
cause significant increases in cytosolic Ca2+ in
transfected HEK cells (average cell volume and capacitance, 4 pl and 20 pF; Figs. 8 and 9). Although our Ca2+ imaging
data are consistent with the findings of others on native and
recombinant
3/
4 receptor measured in neurons, oocytes, and eukaryotic cells, the suppressive effect of Ca2+
on the
3/
4 receptor appears to be more consistent with that observed in HEK cells (Bouisson et al., 1996| |
Footnotes |
|---|
Received November 4, 1998; Accepted March 15, 1999
Supported by National Institutes of Health Grants HL16152 and DA06486.
Send reprint requests to: Dr. Martin Morad, Georgetown University Medical Center, Department of Pharmacology, 3900 Reservoir Road N.W., Washington, DC 20007. E-mail: moradm{at}gunet.georgetown.edu
| |
Abbreviations |
|---|
nAChR, nicotinic acetylcholine receptor; Ach, acetylcholine; HEK, human embryonic kidney; d-tc, d-tubocuraine.
| |
References |
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