Department of Pharmacology, Georgetown University School of
Medicine, Washington, D.C.
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Introduction |
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 |
Materials.
Stably transfected HEK 293 (ATCC CRL
1573)-expressing 3/4 neuronal nAChRs (cell line designation:
KX34R2 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 |
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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Fig. 1.
Activation of currents through the 3/4
receptor channel by different agonists. Currents were recorded at a
constant holding potential of 60 mV with 30 s between each drug
exposure. The traces are typical recordings from different cells.
Notice that epibatidine is effective in a very low (nM) concentration
(C).
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Fig. 2.
Comparison of nicotinic currents activated by
nicotine and cytisine. The sample records show typical currents
activated at 80 mV in the same cell by 50 µM nicotine (A) or
cytisine (B). C, average responses in eight such experiments where the
peak and final (500 ms) currents (arrows in A and B) were normalized
relative to the peak nicotine-induced current. D, dose-response curves
for the peak currents induced by nicotine ( ) and cytisine ( ) at
80 mV. Before curve fitting, the currents induced by the two
compounds were normalized relative to the current induced by 100 µM.
After curve fitting with least-squares determination of
EC50, the data were normalized again to give a maximum
response of one. Each symbol is labeled with a number indicating the
number of cells tested and a vertical error bar indicating the S.E.M.
The continuous curves represent a fit to the Hill equation with a
cooperativity factor close to 2 (see text).
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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.
The differential potency was tested by measuring the responses to 50 µM of both compounds in each of eight cells (Fig. 2, A-C). The
results showed that the peak current activated by nicotine was about
twice as large as that activated by cytisine. Consulting the
dose-response curves (Fig. 2D), this is consistent with the notion of
equal efficacy. Notice, however, that the rate of desensitization was
consistently larger for nicotine than for cytisine, so that the
currents after 500 ms of drug exposure were almost identical (Fig. 2C).
This suggests that the relative potency and efficacy of nicotine and
cytisine may depend on the speed of drug application and the timing of
current or flux measurements.
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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Fig. 3.
Voltage dependence and time course of currents evoked
by different concentrations of nicotine (A), cytisine (B), and
epibatidine (C). Each panel is based on results from a single
representative cell and shows current voltage relations of the maximal
currents produced by the different drug concentrations. The box at the
bottom of each panel indicates the drug concentration corresponding to
each symbol. Notice that 1000 nM epibatidine ( , C) gives less
current than 100 nM ( , C). Each of the three insets in each panel
corresponds to a specific drug concentration and shows the current
traces recorded at different potentials, as labeled.
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Significant outward current was activated by nicotine or cytisine at
potentials positive to +80 mV especially when using 200 to 1000 µM
agonist concentrations (Fig. 3B). The voltage dependence of the
epibatidine- and cytisine-induced current was similar to that of
nicotine (Fig. 3A), i.e., large inward currents were measured at
potentials negative to 0 mV with little or no current activated between
0 and 40 mV (Fig. 3, B and C). The most obvious difference between the
epibatidine and nicotine or cytisine, in addition to the range of
effective concentrations, was the off-rate of epibatidine current upon
removal of the drug. Often at higher concentrations of epibatidine (1 µM), the activated current was significantly smaller than those
generated by 100 nM epibatidine.
Intracellular magnesium has been proposed to regulate the rectification
of the nicotinic current (Mathie et al., 1990; Albuquerque et al.,
1997; Forster and Bertrand, 1995; Bonfante-Cabarcas et al., 1996). We
also examined the effect of
[Mg2+]i on the voltage
dependence of the nicotine-activated current, but found no significant
change in the rectification or the voltage dependence of the current
even when cells were dialyzed for 10 to 15 min with
Mg2+-free intracellular solutions containing 2 mM
of the chelator EDTA (data not shown). Similarly, removal of
extracellular Mg2+ had no measurable effects on
the magnitude or the inward rectifying properties of the current. A
similar insensitivity to Mg2+ was reported in
4/2-expressing HEK cells (Buisson et al., 1996).
Decay 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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Fig. 4.
Desensitization of the nicotine-induced current. The
original records show typical currents evoked at different potentials
(120 mV, 80 mV, 40 mV, and 0 mV) by different concentrations of
nicotine (30 µM, 100 µM, and 1 mM) in different cells with slow (A)
or fast (C) desensitization. B, concentration dependence of the time
constant of desensitization in cells with slowly decaying currents
measured at 120 mV (), 80 mV (), and 40 mV (), and in
cells with rapidly decaying currents measured at 80 mV (). Each
symbol is labeled with number of experiments and a vertical error
bar.
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At high nicotine concentrations, the decay kinetics of current may also
reflect, in part, the block of the open channel by the agonist (Sine
and Steinbach, 1984). Consistent with this idea, the rapid removal of 1 mM nicotine or cytisine were often accompanied by the activation of a
"rebound" current, which may represent the rapid unblock of the
channel by the agonist (Fig. 4A, top right; see also Fig. 7A). The
rebound current was most prominent at voltages negative to 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)
using 1.0 to 10 mM ACh.
In some cells we observed a 10-fold faster rate of desensitization
(Fig. 4C), which was also accelerated by increasing nicotine concentrations ( 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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Fig. 5.
Block of nicotine-induced current by mecamylamine. A,
reversible suppression of nicotine-induced current at 120 mV by 1 µM mecamylamine. B, voltage dependence of nicotine-induced current in
the absence () and presence () of mecamylamine. C, average effect
of mecamylamine effect (A) in four cells (at 120 mV). D, voltage
dependence of mecamylamine block.
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Similarly, d-tc (50 µM) when coapplied with 30 µM
nicotine blocked the current rapidly (Fig.
6). Concentrations of d-tc
smaller than 10 µM, when coapplied with nicotine, had little or no
effect on the current activated by nicotine (n = 3, data not shown). The onset of d-tc block, similar to
mecamylamine, was fast and consistent with channel-blocking properties
of these drugs (Fig. 6A).
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Fig. 6.
Block of nicotine-induced current by
d-tc. A, reversible suppression of nicotine-induced
current by 50 µM d-tc. B, average effect
d-tc effect (A) in three cells.
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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.
Figure 7A shows how the current through
the 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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Fig. 7.
Modulation of the nicotine-induced current by
extracellular calcium. A, sample records showing the currents induced
by 50 µM nicotine when added to the standard Tyrode's solution with
2 mM Ca2+ (left) and when administered together with 10 mM
Ca2+. The traces are labeled with the membrane potential in
mV. B, effect of extracellular Ca2+ on the current induced
by 20 (), 50 (), or 200 () µM nicotine. Currents were
measured as the peak value at 80 mV relative to the average value of
bracketing recordings at 2 mM [Ca2+]o. The
number next to each data point indicates the number of cells examined.
C, average effect of 10 mM Ca2+ on the voltage relations of
the currents induced by 50 µM nicotine. The currents in each
experiment were measured as the average over a 60-ms interval (from
60-120 ms after application of nicotine) and was normalized relative
to the current measured at 80 mV in control solution with 2 mM
Ca2+. D, modulating effect of 10 mM Ca2+ on the
current evoked by nicotine at different potentials.
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To determine more directly whether activation of nicotine-induced
current results in rise of
[Ca2+]i, we measured
confocal images of Ca2+ in Fluo 3-dialyzed
voltage clamped cells. Figure 8A shows
simultaneous traces of nicotine-activated current and intracellular
Ca2+ in response to rapid application of 100 µM
nicotine recorded at indicated locations in the cell. Cytosolic
Ca2+ appears to rise more rapidly at peripheral
and flatter segments of the cell (Fig. 8B, open symbols; see also Fig.
9) than at the thicker midsection of the
cell (Fig. 9B, closed symbols) even though the thicker segments seem to
achieve higher "apparent" Ca2+
concentrations. Intracellular Ca2+ (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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Fig. 8.
Nicotine-induced Ca2+ signals. A, time
course of the current evoked by exposure to 100 µM nicotine (top) and
the normalized change in fluorescence intensity (F/F) measured in
different regions of the cell. Each data point was measured as the
average during four frames and is labeled with a symbol corresponding
to a region in (B). The fluorescence image in (B) was measured
differentially (F) as the rise in fluorescence at the peak of the
response (1-2 s) relative to signal at rest (0-0.5 s). C, compares
the fluorescence signal (F/F) to the integral of the
nicotine-induced current (charge) in five cells indicated with symbols
of different shapes. Open symbols represent measurements performed in
three cells in the presence of 5 µM mecamylamine. The holding
potential in different cells ranged between 60 and 100 mV.
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Fig. 9.
Ca2+ signals evoked by permeation of
Na+ (B) or K+ (C) through the nAChR channel. A,
shows two sets of sample frames measured with confocal microscopy as
nicotinic currents were activated with either Na+ or
K+ as charge carrier. Each of the shown frames was obtained
by averaging four frames recorded at 30 Hz and dividing by the average
of 16 frames recorded before exposure to 100 µM nicotine. The
graphs show the nicotinic currents and Ca2+ signals
recorded in control solution with Na+ as charge carrier (B)
and after replacement of extracellular Na+ with
K+ (C). () correspond to the average signal from cell
body portions of the cell whereas () correspond to average
fluorescence over the three major protrusions as defined by black lines
in the sample frames. The Ca2+ signals (F/F) in the
graphs were obtained by averaging four frames for each point and
integrating over the relevant areas of the cell before normalization.
Arrows indicate the timing of the sample frames.
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The experiment illustrated in Fig. 9 was performed to test the
possibility that the influx and intracellular accumulation of
Na+ might mediate the rise in cytosolic
Ca2+ via transport of Ca2+
on the Na+-Ca2+ exchanger.
Such a mechanism might be accentuated in flatter and narrower parts of
the cell where Na+ as well as
Ca2+ could readily build up as a result of large
agonist-activated current. We therefore compared the
Ca2+ signals measured at 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 |
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; Maconochie and
Steinbach, 1998). The bell-shaped dose-response relation was also seen
in the 3/4-expressing HEK cells in our laboratory using
Rb+ efflux to measure channel function (Xiao et
al., 1998). That agonist may serve as a channel blocker at high
concentrations is also consistent with significant acceleration in the
decay kinetics of 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;
Maconochie and Steinbach, 1998).
Comparing the kinetics of decay of nicotine-activated current in
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). Such cell
populations were relatively rare, and their occurrence could not be
attributed to any known culture or experimental condition.
The kinetics of decay of 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., 1997), compared with about 1.0 s in HEK
cells (Fig. 4). Such differences may reflect the role of host cells in
determining the regulatory properties of neuronal nicotinic receptors
(Lester and Dani, 1995).
Ca2+ 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).
Our electrophysiological data on 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). However, detailed examination of the current-voltage relations
(Fig. 7C) showed that elevation of
[Ca2+]o increased the
inward current at 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). Our findings are in part consistent
with the idea that Ca2+ permeates through the
nAChR channel, but in the process briefly occludes the channel, thereby
lowering its single channel conductance. However, such a mechanism
would require that the blocking effect depend on the electrical
potential driving Ca2+ into the channel, somewhat
inconsistent with Fig. 7D where the degree of block appears to be
equivalent at 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; Vernino et al., 1992), but is not easily reconciled with the
complete suppression of inward current after replacement of all
Na+ with Ca2+ (Fig. 7B). We
also considered whether the rise in cytosolic
Ca2+ occurs indirectly through a voltage-gated
Ca2+ channel or via the
Na+-Ca2+ exchanger. The
persistence of the Ca2+ signals at 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) rather than oocytes (Vernino et al., 1992). It is intriguing to speculate whether the
regulation of Ca2+ permeability of nAChR may, in
part, depend on endogenous presence of other
Ca2+-sensitive ion transporters or subunit
assembly of the receptor in the host cells (Lewis et al., 1997; Cooper
and Miller, 1997).
nAChR, nicotinic acetylcholine receptor;
Ach, acetylcholine;
HEK, human embryonic kidney;
d-tc, d-tubocuraine.
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4 Neuronal Nicotinic Acetylcholine Receptor
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Summary
Introduction
