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The Journal of Neuroscience, June 1, 1998, 18(11):4050-4062
Neuronal Nicotinic Acetylcholine Receptors Are Blocked by
Intracellular Spermine in a Voltage-Dependent Manner
Ali Pejmun
Haghighi and
Ellis
Cooper
Department of Physiology, McGill University, Montréal,
Québec, Canada H3G 1Y6
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ABSTRACT |
A common feature of neuronal nicotinic acetylcholine receptors
(nAChRs) is that they conduct inward current at negative membrane potentials but little outward current at positive membrane potentials, a property referred to as inward rectification. Physiologically, inward
rectification serves important functions, and the main goal of our
study was to investigate the mechanisms underlying the rectification of
these receptors. We examined recombinant  3 4 and 42
neuronal nAChR subtypes expressed in Xenopus oocytes and
native nAChRs expressed on superior cervical ganglion (SCG) neurons.
Whole-cell ACh-evoked currents recorded from these receptors exhibited
strong inward rectification. In contrast, we showed that single-channel
currents from these neuronal nAChRs measured in outside-out patches
outwardly rectify. On the basis of recent findings that spermine, a
ubiquitous intracellular polyamine, confers rectification to glutamate
receptors and inwardly rectifying potassium channels, we investigated
whether spermine causes neuronal nAChRs to inwardly rectify. When
spermine was added to the patch electrode in outside-out recordings, it
caused a concentration- and voltage-dependent block of ACh-evoked
single-channel currents. Using these single-channel data and
physiological concentrations of intracellular spermine, we could
account for the inward rectification of macroscopic whole-cell
ACh-evoked conductance-voltage relationships. Therefore, we conclude
that the voltage-dependent block by intracellular spermine underlies
inward rectification of neuronal nAChRs. We also found that
extracellular spermine blocks both 34 and
42 receptors; this finding points to a
mechanism whereby increases in extracellular spermine, perhaps during
pathological conditions, could selectively block these receptors.
Key words:
neuronal nAChRs; sympathetic neurons; inward
rectification; single-channel currents; spermine; voltage-dependent
block; presynaptic modulation
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INTRODUCTION |
Neuronal nicotinic acetylcholine
receptors (nAChRs) are widespread in the nervous system where they
function as postsynaptic receptors to excite neurons or as presynaptic
receptors to modulate neurotransmitter release (Sargent, 1993 ; Role and
Berg, 1996). Structurally, neuronal nAChRs are pentamers (Anand et al.,
1991; Cooper et al., 1991); however, the precise subunit composition of
functional nAChRs on a given neuron has not been fully resolved (McGehee and Role, 1995). In the peripheral nervous system (PNS), 3 and 4 are the most abundant transcripts
(Boyd et al., 1988; Couturier et al., 1990; Mandelzys et al., 1994),
and most nAChRs are located postsynaptically at synapses formed between
cholinergic preganglionic nerve terminals and postganglionic autonomic
neurons. In the CNS, 4 and 2 transcripts
are the most abundant (Wada et al., 1989; Flores et al., 1992), and
many nAChRs appear to be located on presynaptic nerve terminals
(McGehee et al., 1995; McGehee and Role, 1996). The recent finding that
a missense mutation in the 4 gene underlies autosomal
dominant nocturnal frontal lobe epilepsy highlights the importance of
neuronal nAChRs for normal CNS function (Steinlein et al., 1995;
Weiland et al., 1996).
One striking feature common to all functional neuronal nAChRs,
regardless of whether they are expressed on neurons or heterologously in non-neuronal cells, is the strong inward rectification that results
from a progressive reduction in channel conductance as the membrane
potential depolarizes over the physiological range (Bertrand et al.,
1990; Mathie et al., 1990; Ifune and Steinbach, 1991, 1992; Sands and
Barish, 1992). Physiologically, this inward rectification serves
important functions. For presynaptic receptors, inward rectification
ensures that the receptors will not depress transmitter release by
decreasing the input resistance of the terminal. For postsynaptic
receptors, fluctuations in membrane potential near rest change the
conductance of the receptors and consequently affect synaptic
effectiveness. The factors that cause neuronal nAChRs to rectify have
not been fully characterized. If rectification is not constant, then
changes in rectification can alter synaptic efficacy.
Previous studies suggest that both intrinsic channel properties and
block by intracellular Mg2+ contribute to inward
rectification of neuronal nAChR channels (Mathie et al., 1990; Ifune
and Steinbach, 1991; Sands and Barish, 1992). However, these factors
only partially account for the rectification, suggesting that
unidentified factors are involved (Ifune and Steinbach, 1993; Forester
and Bertrand, 1995). Interestingly, inward rectification of whole-cell
ACh-evoked currents in rat phaeochromocytoma (PC12) cells was reduced
when cells were extensively dialyzed with Na2ATP (Sands and
Barish, 1992; Ifune and Steinbach, 1993). ATP is known to effectively
chelate spermine, a ubiquitous polyamine (Watanabe et al., 1991).
Therefore, it is conceivable that intracellular free spermine
contributes to the inward rectification of neuronal nAChRs, as
demonstrated for inward rectifier potassium channels and glutamate
receptors (Ficker et al., 1994; Bowie and Mayer, 1995; Fakler et al.,
1995). The objective of our study was to examine whether intracellular
spermine produces inward rectification of neuronal nAChRs. We studied
42 and 34
receptors expressed in Xenopus oocytes, as well as native
nAChRs expressed on rat superior cervical ganglion (SCG) neurons. Our
results suggest that inward rectification of neuronal nAChRs can result
from a voltage-dependent block by intracellular spermine.
Parts of these results have been reported in abstract form (Haghighi
and Cooper, 1997).
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MATERIALS AND METHODS |
Preparation of SCG neurons
SCG ganglia were dissected from postnatal day 18-26 Sprague
Dawley rats (Charles River) and dissociated mechanically and
enzymatically as described previously (McFarlane and Cooper, 1992).
Briefly, the ganglia were dissected under sterile conditions from
animals killed by cervical dislocation. The ganglia were dissociated at 37°C either in collagenase (1 mg/ml, type I; Sigma, St. Louis, MO)
for 15 min followed by dispase (2.4 mg/ml, grade II; Boehringer Mannheim, Indianapolis, IN) for 30-45 min or in an enzyme mixture containing trypsin (1 mg/ml; Sigma), deoxyribonuclease (5 µg/ml; Sigma) and bovine serum albumin (6 mg/ml; Sigma) (Mathie et al., 1990)
for 45 min. All enzymes were dissolved in HBSS (without Ca2+ or Mg2+). The ganglia were
gently triturated after the enzyme treatment using a fire-polished
Pasteur pipette until they were completely dissociated. Dissociated
neurons were washed with L-15 medium supplemented with 10% horse serum
and plated onto laminin-coated (40 gm/ml; overnight at 4°C; gift of
Dr. S. Carbonetto, McGill University) Aclar coverslips (Allied
Chemicals, Clifton, NJ) in modified Petri dishes. The neurons were
incubated in 1.5 ml of L-15 medium supplemented with 5% rat serum,
vitamins, cofactors, penicillin, streptomycin, and sodium bicarbonate
as described previously (Hawrot and Patterson, 1979). The media was
also supplemented with nerve growth factor (2.5S NGF, 25 ng/ml; gift of
Dr. S. Carbonetto). The cultures were maintained at 37°C in a
humidified incubator with an atmosphere of 5% CO2 and 95%
air. Neurons were used for electrophysiology within 48 hr (often within
24 hr) after plating.
Preparation and nuclear injection of oocytes
Xenopus oocytes were defolliculated and prepared as
described by Bertrand et al. (1991). We injected 1-3 ng of pairwise
combinations of cDNAs coding for neuronal nAChR subunits
4 and 2, 4 and 2E260, or 3 and 4 into the
nucleus of oocytes. In 2E260, the lysine (K) residue at
the position 260 (part of the extracellular ring of charged residues)
has been substituted with an glutamic acid residue (E), which increases
the single-channel conductance (Cooper et al., 1991). Rat
3 and 4 cDNAs were cloned into the pCDNA1
expression vector (Invitrogen, San Diego, CA), and chick 4, 2, and
2E260 were cloned into derivatives of pSV2.cat
expression vectors (Bertrand et al., 1991). Oocytes were incubated at
19°C for 48-72 hr before recording. For single-channel experiments, the vitelline membrane surrounding the oocytes was removed.
Electrophysiology
Whole-cell recordings from oocytes. To measure the
macroscopic ACh-evoked currents in oocytes, we used two-electrode
voltage-clamp techniques (Bertrand et al., 1991). These experiments
were performed at room temperature (22-24°C) using a standard
voltage-clamp amplifier (built by Mr. A. Sherman, McGill University).
During the recordings, oocytes were perfused with control perfusion
solution or agonist solutions at 10-20 ml/min; the switch from one
solution to another was performed manually. Currents were sampled at
100-350 Hz on-line with a 386-based PC computer (AT class running at
33 MHz and a 64 K cache and A/D card; Omega, Stamford, CT). The program
PATCHKIT (Alembic Software, Montréal) was used for stimulation
and data acquisition. Recording electrodes had tip diameters of 10-15
µm and were filled with 3 M KCl. Only oocytes that gave
rise to large inward currents (>1 µA in response to 1 µM ACh for 42-expressing oocytes or 10 µM ACh for
34-expressing oocytes when
voltage-clamped at  60 mV) were used for single-channel recordings.
External perfusing solution contained 96 mM NaCl, 2 mM KCl, 1 mM
NaH2PO4, 1 mM
BaCl2, 10 mM HEPES, and 1 µM atropine; pH was adjusted with NaOH to 7.4-7.5. Spermine (Sigma) was dissolved in sterile water, and aliquots were kept
frozen at 20°C.
Single-channel recordings from oocytes. Outside-out
recordings were performed using a List EPC-7 amplifier at room
temperature (22-24°C) (Hamill et al., 1981). Pipette resistance
ranged from 5 to 10 M for outside-out recordings, and electrodes
were coated with Sylgard (Dow Corning, Corning, NY). Recordings were
obtained in the continuous presence of ACh (0.1-0.2 µM)
in the recording bath. ACh-evoked single-channel activity gradually
diminished in 2-5 min after excision of the patch. Signals were
digitized with a PCM (501, Sony, Tokyo, Japan) and stored on VCR tapes. For off-line analysis, the stored signals were first filtered at 1.5-2
kHz with an eight-pole Bessel filter (Frequency Devices) and sampled at
10-20 kHz using a 386-based PC computer. The program PATCHKIT was used
for stimulation and data acquisition. External solution contained 100 mM KCl, 1 mM CaCl2, 10 HEPES, and 1 µM atropine, and pH was adjusted to 7.4 with
KOH. Recording electrodes contained 80 mM KF, 20 mM potassium acetate, 10 mM HEPES, and 10 mM EGTA; pH was adjusted to 7.4 with KOH.
Whole-cell recordings from SCG neurons. Whole-cell
patch-clamp recordings on SCG neurons were performed at room
temperature (22-24°C) using a List EPC-7 amplifier (Hamill et al.,
1981). Throughout the recordings neurons were perfused with the
external solution at a rate of 1 ml/min, and agonists were applied by
pressure ejection from pipettes with tip diameters of 20-30 µm
(Mandelzys et al., 1995). Currents were filtered at 1.5 kHz with an
eight-pole Bessel filter (Frequency Devices), sampled at 2.5-5 kHz,
and displayed and stored on-line with a 386-based PC computer. The
program PATCHKIT was used for stimulation and data acquisition. The
resistance of patch pipettes ranged from 2 to 6 M, and 50-60% of
the series resistance was compensated. External perfusing solution
contained 140 mM NaCl, 5.4 mM KCl, 0.33 mM NaH2PO4, 0.44 mM KH2PO4, 2.8 mM CaCl2, 10 mM HEPES, 5.6 mM glucose, 2 mM glutamine, 0.5-1
µM TTX (Sigma), and 1 µM atropine; pH was
adjusted to 7.4. ACh (acetylcholine iodide; Sigma) was dissolved in the
same external perfusing solution (100 µM). Recording
electrodes were filled with intracellular solution containing 70 mM KF, 65 mM potassium acetate, 5 mM NaCl, 1 mM MgCl2, 10 mM EGTA, and 10 mM HEPES; pH was adjusted to
7.4 with KOH.
Single-channel recordings from SCG neurons. Outside-out
recordings were performed on SCG neurons as described above for
oocytes. Agonists were applied by a manual switch through a
micropipette, and neurons were washed with control solutions between
each recording. Recording electrodes were fire-polished after they were
coated with Sylgard. SCG outside-out patches also showed a rapid
rundown of single-channel activity (2-4 min) in all cases. External
solution was identical to that described for whole-cell experiments
except that the ACh concentrations were 5-20 µM.
Recording electrodes contained 70 mM Cs-gluconate, 70 mM CsF, 1 mM MgCl2, 10 mM HEPES, and 10 EGTA; pH was adjusted to 7.4 with
CsOH.
Voltage-clamp protocols. For I-V
curves we measured ACh-evoked currents either by holding the membrane
potentials (Vm) at different levels or in
response to voltage ramps. Voltage-ramp protocols were as follows:
whole-cell oocytes, 333 mV/sec; whole-cell neurons, 333 or 200 mV/sec;
outside-out recordings, 1 V/sec, 333 mV/sec, or 200 mV/sec. We
also used voltage-step protocols in which we stepped the holding
potential rapidly between 40, 50, and 60 mV and held at each potential
for 1 sec.
Analysis
Whole-cell and single-channel ramp I-V
curves were obtained by subtracting the current in the absence of
agonist from that in the presence of agonist. All
I-V curves were fit by eye to a polynomial
function using Origin 4.1 graphics software (MICROCAL Software). To
measure the amplitude of single-channel currents, we used either
all-points histograms of open and closed distributions, or we measured
the amplitude of the channel openings individually using PATCHKIT and
then plotted values (>50 openings) on a histogram. Histograms were fit
by gaussian curves using Origin 4.1 graphics software. Some outside-out
patches, especially from SCG neurons, gave rise to a nonspecific
steady-state conductance at potentials above +20 mV, which made the
clear detection of ACh-evoked single-channel openings difficult.
To measure the relative open-channel probability
(popen), we used single-channel
recordings in response to 1 sec voltage steps between ± 40, ± 50, and ± 60 mV. At each Vm, we
integrated the area under the all-points amplitude histograms
corresponding to closed and open states of the receptors and
then divided the area of the open state by the total area. Then we
calculated the relative popen for ± 40, ± 50, and ± 60 mV by dividing the popen at
the positive Vm by that at the corresponding
negative Vm. The conductance (G) was determined from G = I/(Vm Erev).
We used a derivation of the Woodhull (1973) equation (see Results) to
fit the G-V relationships. All fits were
performed using Origin 4.1 based on a Levenberg-Marquardt algorithm,
and the best fit was achieved by  2 minimization.
Statistical significance was examined using ANOVA.
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RESULTS |
Inward rectification of macroscopic ACh currents
ACh-evoked currents through nicotinic receptors on neurons are
carried by small monovalent and divalent cations. A property common to
all functional neuronal nicotinic receptors is that they conduct inward
current when the membrane potential (Vm)
is negative, but pass little outward current when
Vm is positive. To observe the rectification on
neonatal rat SCG neurons, we applied voltage ramps to change
Vm from 60 mV to +50 mV at a rate of 333 mV/sec before and during ACh application (Fig.
1A). We obtained the
current-voltage (I-V) curve by
subtracting the control current from the current during the ACh
application (Fig. 1B). The small amount of
desensitization of the ACh-evoked current had minimal effect on the
shape of the I-V curve. The ratio of the
whole-cell ACh-evoked current at +50 mV to that at 60 mV is 0.01 ± 0.008 (n = 10). Similar rectification is observed
when Vm is held steady at different potentials
during ACh application (data not shown). Comparable inward
rectification has been reported previously for ACh-evoked currents in
adult rat sympathetic neurons (Mathie et al., 1990, 1991), rat adrenal
chromaffin cells (Hirano et al., 1987), and rat PC12 cells (Neuhaus and
Cachelin, 1990; Ifune and Steinbach, 1992; Sands and Barish, 1992).
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Figure 1.
Macroscopic ACh-evoked currents recorded from SCG
neurons show strong inward rectification. A, Whole-cell
currents were recorded from an SCG neuron while the membrane potential
was ramped from 60 to +50 mV (333 mV/sec). The top
trace shows the response of the neuron in the absence of
agonist, and the bottom trace shows the response to 100 µM ACh. B, This figure shows the
current-voltage (I-V)
relationship for the neuron in A. The whole-cell
I-V curve was obtained by subtracting
the current in control solution from the current in the presence of
ACh, during the voltage ramp. The I-V
plot shows strong inward rectification.
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Recombinantly expressed neuronal nAChRs also exhibit strong inward
rectification. Figure
2A,B shows an example
of an ACh-evoked I-V curve and the corresponding
chord conductance-voltage (G-V) curve
for 34 receptors expressed in
Xenopus oocytes. Figure 2C,D shows an example of
an ACh-evoked I-V curve and the
G-V curve for 42
receptors expressed in Xenopus oocytes. These figures demonstrate that both 42 and
34 receptors show strong inward rectification, and the conductance of these receptors progressively decreases as Vm depolarizes from 80 mV. We
have also investigated a mutant 42
receptor in which the lysine at position 260 on the 2
subunit has been changed to a glutamic acid; this mutation increases
the single-channel conductance of the 42
receptors (Cooper et al., 1991) (Fig. 3),
and we refer to it as 42E260. Our reason
for using this mutant receptor is that alteration of charged residues
at the extracellular mouth of the pore has been shown to influence
rectification of muscle nAChRs (Imoto et al., 1988). Figure
2E shows an example of an I-V
curve for 42E260 receptors expressed in
Xenopus oocytes. Qualitatively,
42E260 receptors rectify in a manner
similar to wild-type 42 receptors. Similarly, the G-V plot in Figure
2F demonstrates that the conductance of
42E260 receptors decreases progressively
as Vm depolarizes. At very positive potentials
(>60 mV), all three receptors conduct some outward current (data not
shown). The V1/2 values obtained from the
G-V curves are not significantly different for
34, 42, and
42E260 subtypes and for native nAChRs on
SCG neurons; this suggests that their inward rectification properties
are similar.
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Figure 2.
Whole-cell ACh-evoked current-voltage
relationships for recombinant 34,
42, and
42E260 expressed in
Xenopus oocytes show strong inward rectification.
A, C, and E show
whole-cell I-V curves for three oocytes
expressing recombinant 34,
42, and
42E260, respectively. Macroscopic
currents were recorded in response to voltage ramps from 80 to +50 mV
(333 mV/sec) in the absence or presence of ACh (1 µM for
42 and
42E260, and 20 µM for
34), and the net currents were
obtained as explained in Figure 1. Note: the strong inward
rectification of the I-V curve is
similar to that of whole-cell I-V in SCG
neurons (Fig. 1B). B,
D, and F show the corresponding
conductance-voltage relationships (G-V
plot) for 34,
42, and
42E260, respectively. Conductance at each
holding potential is normalized to the conductance at 80 mV.
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Figure 3.
Outside-out single-channel currents from
Xenopus oocytes expressing
34,
42, and
42E260 do not show inward rectification.
A, C, and E show
steady-state single-channel recordings from outside-out patches of
oocytes expressing 34,
42, or
42E260, respectively. These recordings
were performed in the continuous presence of ACh: 100-200
nM for 42 and
42E260 and 1-2 µM for
34. Numbers on the
left of each trace correspond to the holding potential
at which that recording was obtained. The dotted lines
mark the zero current level for each trace. B,
D, and F show the single-channel
I-V plots for
34,
42, or
42E260, respectively. These
I-V plots, in contrast to the
corresponding macroscopic I-V plots
(Fig. 2A,C,E), show a slight outward
rectification. Each point in the plots represents the mean ± SE
of single-channel current amplitudes from four patches for
34, eight patches for
42, and eight patches for
42E260.
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Lack of inward rectification in single nAChRs measured from
outside-out patches of Xenopus oocytes
In contrast to what we observed at the macroscopic level, single
nAChRs measured in outside-out patches conducted current in both
directions equally well through the receptor. Figure 3 shows examples
of single-channel currents from
34,
42, and 42E260 receptors recorded from
outside-out patches held at four different potentials (Fig.
3A,C,E) and the corresponding single-channel I-V curves (Fig. 3B,D,F).
These records were obtained in symmetrical K+
concentrations (100 mM), with K+ being
the main charge carrier. Under these conditions,
34 receptors (Fig. 3A) have a
single-channel conductance of 45 ± 0.87 pS (mean ± SE, at 100
mV) and rectify slightly outward [(I@+60
mV)/(I@60 mV) = 1.2 ± 0.13]. Similarly, both
42 and
42E260 receptors in outside-out patches
conducted current in both directions (Fig. 3C,E). The
single-channel conductance for 42
receptors recorded in symmetrical K+ was 49 ± 0.68 pS and that for 42E260 was 61 ± 0.81 pS. All three receptors showed a smaller single-channel
conductance when extracellular K+ was substituted
with Na+ (Cooper et al., 1991) or
Cs+ (data not shown). The I-V
curve for 42E260 receptors shows less
outward rectification than those for 34
and 42 receptors as a result of
introducing of negative charges to the extracellular ring of the pore
(Imoto et al., 1988).
The results in Figure 3 indicate that inward rectification of
macroscopic ACh-evoked currents is not because neuronal nACh receptors
cannot pass outward currents. Similar results have been reported for
nAChRs on adult rat sympathetic neurons (Mathie et al., 1990, 1991),
rat adrenal chromaffin cells (Hirano et al., 1987), and rat PC12 cells
(Neuhaus and Cachelin, 1990; Ifune and Steinbach, 1991, 1992; Sands and
Barish, 1992).
We investigated whether the rectification of macroscopic ACh-evoked
currents occurs because these receptors have a decreased open channel
probability (popen) at depolarized
potentials. For these experiments, we recorded single-channel currents
from 42 and
42E260 receptors in outside-out patches
while repeatedly stepping Vm between ±40, ±50
(Fig. 4A), and ±60 mV.
The results indicate that there is a 25-30% reduction in
popen at +40, +50, and +60 potentials compared
with popen at the corresponding negative potentials:
popen(+40)/popen( 40) = 0.72 ± 0.3;
popen(+50)/popen(50) = 0.75 ± 0.2;
popen(+60)/popen(60) = 0.71 ± 0.4 (n = 3 patches, 10-20 steps per
patch). This reduction of popen at depolarized
potentials, however, is too small to account for the inward
rectification observed at the whole-cell level.
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Figure 4.
Voltage-dependence at the single-channel level
cannot account for the macroscopic inward rectification.
A, Representative trace showing
42 single-channel recordings from an
outside-out patch while Vm was stepped
between 50 and +50 mV (1 sec at each
Vm). Dotted lines mark
the zero current level. B, This trace shows currents
recorded from an outside-out macropatch expressing
42 nAChR subtypes. The current was
recorded in response to 200 nM ACh while
Vm was ramped from 85 to +85 mV at 1 V/sec. The I-V curve was obtained by
subtracting the current after run-down of single-channel activity from
the current obtained 30 sec after excision of the patch.
C, This trace shows two superimposed
42 single-channel recordings from an
outside-out patch while the Vm was ramped
from 100 to +90 mV (1 V/sec). These traces were obtained in the
continuous presence of 100 nM ACh. D, This
figure shows single-channel currents from an outside-out patch
expressing 42E260 (ACh = 200 nM) while Vm was ramped from
100 to +90 mV (1 V/sec). For this patch, 1 mM
Mg2+ had been added to the recording pipette. Note:
outward single-channel openings at positive membrane potentials are
smaller than those in control recordings, suggesting a moderate block
by intracellular Mg2+.
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In a few experiments, we also measured ACh-evoked currents from
macropatches in outside-out configuration while ramping the membrane
potential (Fig. 4B). Consistent with what we observed for popen in the experiments above, the
ACh-evoked currents from these macropatches exhibited only a small
degree of rectification.
To test whether fast changes in the membrane potential can affect the
opening of the receptors, we recorded ACh-evoked single channels from
outside-out patches while rapidly ramping the patch holding potential
from 100 to +90 mV. Figure 4C shows two superimposed 42 single-channel records measured under
these conditions. These records illustrate opening of single-channel
42 receptors while the membrane potential
is rapidly changed from 100 to +90 mV at 1 V/sec. The average
frequency of opening at positive potentials was 20-25% less than that
at negative potentials.
The results of these cell-free single-channel experiments suggest that
inward rectification of the ACh-evoked current is caused by an
intracellular(s) mediator. It had been suggested that intracellular Mg2+ could cause a moderate rectification at
single-channel level through a voltage-dependent blocking mechanism
(Mathie et al., 1990; Ifune and Steinbach, 1991). However, extensive
chelation of intracellular Mg2+ had very little
effect on the rectification properties of the whole-cell currents
(Mathie et al., 1990; Neuhaus and Cachelin, 1990). We found that
including Mg2+ (up to 1 mM) in the
pipette solution in outside-out recordings had little effect on either
the single-channel I-V curves for both
42 and
42E260 receptors (Fig.
4D) or on popen at depolarized potentials (data not shown). These findings confirm previous results that intracellular Mg2+ is not a mediator of inward
rectification of macroscopic ACh-evoked currents.
Intracellular spermine blocks neuronal nAChRs expressed in
Xenopus oocytes
Recent studies have reported that intracellular spermine, an
intracellular polyamine, can block inwardly rectifying
K+ channels and AMPA and kainate receptors in a
voltage-dependent manner (Ficker et al., 1994; Bowie and Mayer, 1995;
Fakler et al., 1995; Kamboj et al., 1995). We investigated whether
spermine also blocks neuronal nAChRs. To test this, we recorded
single-channel currents from 42 and
42E260 receptors in outside-out patches with different concentrations of spermine (100 nM to 100 µM) added to the pipette solution. Figure
5A shows an example of
single-channel currents for 42E260
receptors recorded with 33 µM spermine in the recording
electrode; the presence of 33 µM spermine abolished single-channel current at holding potentials >0 mV. At lower
concentrations (1-3.3 µM), spermine reduced the
amplitude of the single-channel openings (Fig. 5B). The fact
that the outward currents are reduced in size, as seen in Figure
5B, suggests that spermine acts with fast kinetics (Hille,
1992). The single-channel I-V curves for 42 receptors obtained from 14 outside-out
patches in the presence of three different spermine concentrations are
shown in the Figure 5C; for concentrations of spermine  10
µM, outward currents are abolished for
Vm from 0 mV to +50 mV. In addition, comparison of data in control solutions clearly shows that spermine causes some
reduction of the inward current at negative Vm.
The effects of spermine on the single-channel
I-V curves of 42
and 42E260 receptors (n = 12) were similar.
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Figure 5.
Spermine causes inward rectification of ACh-evoked
currents in outside-out patches. A, These traces are
outside-out single-channel recordings (100 nM ACh) at
different holding potentials from an oocyte expressing
42E260 receptors with 33 µM
spermine added to the intracellular pipette solution. Dotted
lines mark the zero current level. B, This
figure shows outside-out recordings (ACh = 100 nM)
from an oocyte expressing 42 receptors
with 1 µM spermine added to the recording electrode; the
outward 42 single-channel currents at +60
mV are reduced by ~40%. Dotted lines mark the first
open channel level for each trace. C, This figure shows
the steady-state single-channel I-V
relationship for 42 receptors in the
absence ( ; n = 12) or in the presence of 1 µM ( ; n = 4), 10 µM
( ; n = 6), or 100 µM ( ;
n = 4) spermine in the recording electrode.
I-V plots show progressive inward
rectification of single-channel current with increasing concentrations
of intracellular spermine. Solid lines are polynomial
fits.
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The effects of spermine are voltage- and concentration-dependent.
Figure 6A shows the
single-channel G-V relationship for 42 receptors with increasing spermine
concentrations. At each Vm, every point
represents the single-channel conductance in the presence of spermine
as a fraction of the control; all points are normalized to their
single-channel conductance at 100 mV. In control solutions without
spermine, the single-channel conductance shows slight outward
rectification. However, with spermine in the recording electrode the
single-channel conductance decreases with depolarization in a sigmoidal
manner and falls to near zero at positive potentials (except for
spermine concentrations <3.3 µM). Higher concentrations
of spermine result in parallel leftward shifts of the
G-V curves. With spermine concentrations >3.3
µM, the sigmoidal G-V curve at the
single-channel level resembled the G-V curves
measured at the macroscopic level (compare with Fig. 2), suggesting
that the voltage-dependent block by intracellular spermine may underlie
the inward rectification observed for the whole-cell ACh-evoked
currents. At membrane potentials positive to +50 mV (+20 mV for <3.3
µM spermine), we observed an increase in the conductance.
Small outward currents were also apparent at these potentials at the
whole-cell level (data not shown).
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Figure 6.
Analysis of voltage- and concentration-dependent
block by intracellular spermine. A, This figure shows
single-channel G-V relationships for
42 in the absence or the presence of 1, 3.3, 10, 33, 50, or 100 µM spermine in the recording
electrode. Single-channel conductance in control patches increased
with depolarization (n = 14), whereas
single-channel conductance in the presence of spermine
progressively decreased with depolarization. Increasing the
concentration of spermine causes a leftward shift of the
G-V relationship. Solid
lines are fits to data points using Equation 1. Points
represent the single-channel conductance in the presence of spermine as
a fraction of the control conductance for each holding potential; all
points are normalized to the conductance at 100 mV. Data are obtained
from three to six outside-out patches for each spermine concentration
(mean ± SE). Dotted lines are polynomial fits to
the data. B, This figure shows the
concentration-response relationship for spermine at different holding
potentials. Affinity of spermine for 42
receptor is increased with depolarization. Data points are derived from
A, and solid lines are fits using
Equation 1. The dotted line is the theoretical fit at 0 mV predicted by Equation 1. C, This figure shows a
representative G-V plot of the
macroscopic ACh-evoked current recorded from an oocyte expressing
42 receptors. We fit this
G-V curve with Equation 1, using the
Kd(0) and z values
obtained from single-channel analysis (solid line).
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To analyze the effects of spermine at the single-channel level, we used
a derivation of the Woodhull model (Eq. 1) for voltage-dependent ion
channel block (Woodhull, 1973; Johnson and Ascher, 1990):
![G/G<SUB><UP>max</UP></SUB>=1/{1+[S]/K<SUB><UP>d</UP></SUB>}](/content/vol18/issue11/fulltext/4050/img001.gif)
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(1)
|
where
and [S] is the internal spermine concentration,
Kd is the dissociation constant at a given
Vm, Kd(0) is the
dissociation constant at 0 mV, z is the valence of spermine,
is the fraction of the membrane electrical field sensed by spermine
from the intracellular side, and R, T, and
F refer to the universal gas constant, the absolute
temperature, and the Faraday constant, respectively.
The solid lines in Figure 6A show that Equation 1
provides a good description of the single-channel
G-V curves. Assuming the valence of spermine,
z, to be 3.8 at physiological pH (Bowie and Mayer, 1995),
this analysis estimates Kd(0) and to be
3.64 ± 0.25 µM and 51%, respectively
(z = 1.93 ± 0.3), and is based on data from 25 outside-out patches expressing 42
receptors at eight different concentrations of spermine. Analysis of
the spermine block of 42E260 receptors
gave very similar results: Kd(0)= 3.58 ± 0.20 and z = 1.85. Figure 6B shows the
effects of Vm on the affinity of the receptor
for spermine (Kd). Within a range of
Vm from 80 mV to +30 mV, the
concentration-dependent block by spermine was well described by a
single binding isotherm (Fig. 6, solid lines), with the
Kd decreasing with increasing depolarization:
1399, 316, 71.4, 16.1, 3.64, and 0.39 µM for 80, 60,
40, 20, 0, and +30 mV, respectively. These values are comparable to
those for recombinantly expressed AMPA and kainate receptors (Bowie and
Mayer, 1995).
Using the values for z and Kd(0)
from the single-channel measurements above, we fit the
G-V curves for macroscopic ACh-evoked currents
for 42 receptors using Equation 1. Figure
6C shows that Equation 1 describes the data well assuming an
intracellular spermine concentration [S] of 82 µM (n = 8). It should be noted that this
fit does not take into account the possible contribution of other
polyamines such as spermidine and putrescine or the moderate voltage-dependent decrease in single-channel
popen.
Spermine blocks native neuronal nAChRs on sympathetic neurons
Our results on recombinant receptors indicated that intracellular
spermine blocks neuronal nAChRs in a voltage-dependent manner, thereby
producing inward rectification of the ACh-evoked currents. To determine
whether spermine has a similar action on native receptors expressed on
neurons, we examined the effects of intracellular spermine on nAChRs
expressed by sympathetic neurons. Figure
7A shows that single nAChR
currents measured in cell-free outside-out patches flow in both
directions equally well, as has been reported previously (Mathie et
al., 1990). The single-channel conductance measured in outside-out
patches from SCG neurons was 32.5 ± 1.35 pS at 60 mV
(n = 12). Spermine (100 µM) added to the
recording electrode, completely abolished single-channel current at
Vm >0 mV (Fig. 7B). These results
suggest that intracellular spermine in sympathetic neurons can block
nAChRs from the inside and produce inward rectification of the
ACh-evoked currents. We plotted the single-channel
I-V curves using data from seven control
outside-out patches and four outside-out patches in the presence of
spermine (Fig. 7C). The control I-V
curve shows slight outward rectification, whereas the single-channel
I-V curve in the presence of 100 µM spermine exhibits a strong inward rectification,
similar to that seen at the macroscopic level (compare with Fig.
1B). We also measured ACh-evoked currents in
outside-out macropatches. As shown in Figure 7D, in the
presence of spermine (100 µM) the ACh-evoked current from
a macropatch shows strong inward rectification.
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Figure 7.
Intracellular spermine blocks native SCG nAChRs at
the single-channel level. A, This figure shows
single-channel recordings obtained from an SCG neuron in an outside-out
patch; native SCG nAChRs show both inward and outward currents in
response to 20 µM ACh. Dotted lines mark
the zero current and the first open state. B,
Outside-out single-channel recordings from another SCG neuron with 100 µM spermine added to the recording electrode. Spermine
(100 µM) completely blocked the outward single-channel
openings and reduced the amplitude of the inward openings ([ACh] = 20 µM). At positive potentials, dotted lines
mark the baseline, and at negative potentials, dotted
lines mark the baseline and the first open state.
C, This figure shows ACh-evoked single-channel
I-V curves obtained from outside-out
patches from SCG neurons. The control
I-V curve ( ; n = 7) exhibits slight outward rectification, similar to recombinant
neuronal nAChRs (Fig. 3B,D,F). In the presence of
100 µM spermine in the recording electrode (;
n = 4), however, the
I-V relationship exhibits strong inward
rectification. Single-channel amplitudes were measured in both
steady-state and ramp experiments, and points represent mean ± SE. Solid lines are polynomial fits. D,
This figure shows the current in response to 20 µM ACh
recorded from an outside-out macropatch from an SCG neuron in the
presence of 100 µM spermine in the recording electrode.
The current was recorded while Vm was ramped
from 60 to +50 mV (at 1V/sec). The solid line is a
polynomial fit.
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Figure 8A shows the
G-V plot obtained from four outside-out patches
of SCG neurons with 100 µM spermine added to the
recording electrode. Similar to our results for
42 receptors, the block by spermine of
native SCG nAChRs increases as the membrane is depolarized in a
sigmoidal manner. Using Equation 1, we fit this single-channel
G-V curve and estimated
Kd(0) and to be 4.27 ± 0.6 µM and 65%, respectively (with z being
2.48 ± 0.16). These results suggest that spermine has comparable
affinity for 42 receptors and native
nAChRs expressed on SCG neurons.
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Figure 8.
Block by intracellular spermine underlies the
inward rectification of macroscopic ACh-evoked currents in SCG neurons.
A, This figure shows the single-channel
G-V plot for native SCG nAChRs in the
presence of 100 µM spermine. The single-channel
conductance shows a progressive decrease with depolarization. Points
represent the single-channel cord conductance (mean ± SE;
n = 4) in the presence of spermine as a fraction of
the control single-channel conductance at every potential; all values
were normalized to the conductance at 75 mV. The solid
line is the fit to the data using Equation 1. B,
This figure represents the whole-cell
G-V plot obtained from an SCG neuron.
Similar to the single-channel conductance in the presence of spermine,
the macroscopic ACh-evoked conductance in SCG neurons shows a
progressive decrease with depolarization. The solid line
is a fit to these data using Equation 1. This fit was performed using
values for Kd(0) and z
from single-channel analysis (see Results).
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Using the parameters obtained from the single-channel analysis, we
analyzed the macroscopic ACh-evoked currents in SCG neurons. In
Figure 8B, we transformed a whole-cell ACh-evoked
I-V curve into a G-V
curve, assuming a reversal potential of 5 mV (our unpublished
observations) for ACh-evoked currents in SCG neurons. The macroscopic
chord conductance progressively decreases as Vm is depolarized from 65 mV (Fig. 8B). We used
Equation 1 to fit the whole-cell ACh-evoked G-V
curves using Kd(0) and as determined at the
single-channel level (Fig. 8B, solid
line). Results from eight different experiments estimated the
intracellular spermine concentration, [S], to be 51 µM. The exact concentration of free spermine in rat
sympathetic neurons has not been determined, but the value predicted
from Equation 1 is within the range measured for other mammalian cells
(Watanabe et al., 1991) and comparable to that estimated in human
embryonic kidney cells using similar analysis for kainate receptors
(Bowie and Mayer, 1995).
Extracellular spermine blocks neuronal nAChRs
Extracellular polyamines have been shown to block both native and
recombinant AMPA receptors (Robichaud and Boxer, 1993; Washburn and
Dingeldine, 1996). Therefore, we asked whether extracellular spermine
can also block neuronal nAChRs and whether this effect is similar to
the voltage-dependent block produced by intracellular spermine.
Extracellular spermine blocked the inward ACh-evoked currents in
oocytes expressing 42 (Fig.
9A). Co-application of 33 µM spermine with 1 µM ACh in the
extracellular solution resulted in a ~45% block of the macroscopic
ACh-evoked inward current recorded from
42 receptors (Fig. 9A).
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Figure 9.
Extracellular spermine blocks macroscopic
ACh-evoked inward currents. A, This figure shows inward
ACh-evoked currents recorded from an oocyte expressing
42 in the absence and the presence of
extracellular spermine. At 90 mV, 33 µM extracellular
spermine blocks the current in response to 1 µM ACh by
45%. B, Macroscopic I-V
relationship for the oocyte in A is plotted in the
presence and absence of 33 µM extracellular spermine.
Inward rectification is not affected by extracellular spermine.
C, This figure shows the change in the macroscopic
conductance versus the membrane potential in the presence of 33 µM extracellular spermine. The conductance in the
presence of spermine is shown as a fraction of the control conductance
at every membrane potential and then normalized to the conductance at
90 mV. D, This figure shows the inhibition curve for
42 receptors with increasing
concentrations of extracellular spermine. Extracellular spermine blocks
the ACh-evoked current in 42-expressing
oocytes in a concentration-dependent manner. Each spermine
concentration was tested on at least seven oocytes (mean ± SE).
Solid line is a fit for a single binding site isotherm
(see Results).
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To examine the voltage-dependence, we measured the block when ramping
the membrane potential from 90 mV to +50 mV. Figure 9B
shows the ACh-evoked I-V curves for
42 receptors in the absence or presence
of 33 µM extracellular spermine. The form of the typical inward rectification of the I-V curve is not
affected by extracellular spermine. We also plotted the changes in
macroscopic conductance (as a fraction of the control conductance) of
the receptor versus membrane potential in the presence of 33 µM spermine (Fig. 9C). As is apparent from
this G-V plot, the blocking effect of
extracellular spermine shows little voltage-dependence. To test whether
extracellular spermine can act as a competitive antagonist, we examined
the effect of increasing concentrations of ACh on the block by
extracellular spermine. The blocking effect of 50 µM
extracellular spermine on inward currents evoked by either 0.1, 1, or
10 µM ACh in 42-expressing oocytes was not significantly different (n = 5).
We tested the effect of 1 nM to 1 mM
extracellular spermine on ACh-evoked currents in oocytes expressing
34,
42, or
42E260. The inhibition curves for all
three receptors were well described by a single binding site isotherm
according to the equation: I = Imax/(1 + [S]/IC50), where I is the
current measured at any Vm in the presence of
spermine, Imax is the current at the same Vm in the absence of spermine, [S]
is the concentration of extracellular spermine, and IC50 is
the concentration of spermine for half-maximal inhibition. The
extracellular block by spermine was concentration-dependent, with an
IC50 of 40.3 ± 3.8 µM
(n = 7) (Fig. 9D) for
42 receptors. We obtained similar results
for 34 (42.3 ± 4.3 µM; n = 5) and 42E260 (39.5 ± 2.7 µM; n = 6) expressed in oocytes (data not shown).
| |
DISCUSSION |
In this study, we investigated mechanisms involved in the strong
inward rectification of neuronal nAChRs. Our results demonstrate that
spermine blocks neuronal nAChRs with high affinity at depolarized membrane potentials when acting on the cytoplasmic side of the channel.
Given the high concentration of free spermine in neurons, the
voltage-dependent block by intracellular spermine likely underlies inward rectification of neuronal nAChRs.
We examined the effects of spermine on three different subtypes of
neuronal nAChRs: recombinant 34 and
42 receptors expressed in
Xenopus oocytes, and native receptors expressed by SCG
neurons. At the whole-cell level, we demonstrated that the ACh-evoked
currents for all three receptors exhibit strong inward rectification,
consistent with previously published reports (Bertrand et al., 1990;
Mathie et al., 1990; Ifune and Steinbach, 1992; Sands and Barish,
1992). Furthermore, at the single-channel level, we showed that this inward rectification is abolished in cell-free outside-out patches, as
shown for neuronal nAChRs expressed on SCG neurons and PC12 cells
(Mathie et al., 1990; Neuhaus and Cachelin, 1990; Ifune and Steinbach,
1992; Sands and Barish, 1992).
Intracellular spermine has been shown to underlie inward rectification
of glutamate receptors (Bowie and Mayer, 1995) and inwardly rectifying
K+ channels (Fakler et al., 1995); therefore, we
examined the effects of intracellular spermine on neuronal nAChRs. In
outside-out recordings, we found that the addition of spermine
(0.1-100 µM) to the recording pipette produced a
voltage-dependent block of neuronal nAChRs in a concentration-dependent
manner. To analyze the spermine block, we fit the single-channel
G-V curves to a derivation of the Woodhull model
(Woodhull, 1973). According to this model, spermine enters the open
receptor and binds to a site within the membrane electrical field.
Spermine at this site interferes with the movement of small cations
through the channel pore, and the occupancy of this site by spermine is
affected by changes in membrane potential. This analysis gave a
Kd(0) of 3.6 µM and a
z of 1.9 for 42 receptors and a Kd(0) of 4.3 µM and a
z of 2.5 for nAChRs on SCG neurons, suggesting that the
spermine binding site is located at 51 or 65% of the membrane
electrical field, respectively. These values are comparable to those
measured for kainate receptors (Kd(0) = 5.5 µM and z = 2.5) and AMPA receptors
(Kd(0) = 1.5 µM) (Bowie and Mayer,
1995).
Using the Woodhull model with the above parameters, we were able to
describe the G-V relationships for whole-cell
ACh-evoked currents and estimate the free intracellular spermine in
oocytes (82 µM) and SCG neurons (51 µM).
These values are well within the range (50-200 µM) of
physiological free spermine concentrations (Watanabe et al., 1991;
Bowie and Mayer, 1995; Traynelis et al., 1995). Although our analysis
suggests that spermine alone is capable of conferring rectification to
ACh-evoked currents in neurons, it is possible that other polyamines
such as spermidine and putrescine contribute to the action of
spermine.
The model also assumes that spermine does not pass through the channel
(Woodhull, 1973). This model describes our data well for membrane
potentials from 100 mV to +50 mV. However, at membrane potentials
>50 mV (or +20 mV for spermine <3.3 µM), |
Top
Abstract
Introduction
![K<SUB><UP>d</UP></SUB>=K<SUB><UP>d</UP>(<UP>0</UP>)</SUB><UP>exp</UP>{<UP>−</UP>V<SUB><UP>m</UP></SUB>z&dgr;F/[RT]}](/content/vol18/issue11/fulltext/4050/img002.gif)
