Department of Biology, University of California, San Diego, La Jolla, California 92093
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ABSTRACT |
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Liu, Qing-Song and
Darwin K. Berg.
Extracellular Calcium Regulates Responses of Both 3- and
7-Containing Nicotinic Receptors on Chick Ciliary Ganglion
Neurons.
J. Neurophysiol. 82: 1124-1132, 1999.
Neuronal nicotinic receptors are generally
both permeable to calcium and potentiated by it. We have examined acute
calcium regulation of both native
7-containing and the less abundant
3-containing nicotinic receptors on chick ciliary ganglion neurons. Most of the receptors are concentrated on somatic spines tightly overlaid in situ by a large presynaptic calyx. Whole cell patch-clamp recording from dissociated neurons using perforated patch-clamp techniques indicates that the rapidly desensitizing nicotinic response
of
7-containing receptors achieves maximum amplitude in 2 mM
calcium; both lower and higher concentrations of calcium are less
effective. Barium and strontium but not magnesium can substitute for
calcium in potentiating the response. Neither calcium current through
the receptors nor calcium action at intracellular sites is necessary.
These latter conclusions are supported by current-voltage analysis of
the nicotine-induced response, ion substitution experiments, and
internal perfusion of the cells with
1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic
acid (BAPTA) via a conventional patch pipette. Varying the agonist concentration indicates that some of the calcium-dependent enhancement may involve a shift in the dose-response curve for agonist binding, but
much of the effect is also likely to involve increased receptor responsiveness. Blockade of
7-containing receptors with
-bungarotoxin showed that the heteromeric
3-containing nicotinic
receptors also undergo calcium-dependent potentiation. Calcium did not
have a major effect on the desensitization rate of either receptor class but did have a selective effect on the rise time of
7-containing receptors. Analysis of stably transfected cells
expressing an
7 gene construct showed that the calcium potentiation
observed for native receptors did not require neuron-specific
modifications or components and that it could be seen with the natural
agonist acetylcholine. Receptor dependence on extracellular calcium may provide a regulatory mechanism for constraining synaptic signaling, avoiding local depletion of external calcium, and limiting calcium buildup in postsynaptic compartments.
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INTRODUCTION |
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Neuronal nicotinic acetylcholine receptors
(nAChRs) have an usually high relative permeability to calcium
(Fieber and Adams 1991; McGehee and Role
1995
; Mulle et al. 1992a
; Rathouz and
Berg 1994
; Rathouz et al. 1996
; Vernino
et al. 1992
, 1994
). Most pronounced are those
containing the
7 gene product (
7-nAChRs), which have been
estimated to be 6-20 times more permeable to calcium than to sodium
(Bertrand et al. 1993
; Seguela et al.
1993
; Vernino et al. 1994
). The relative
abundance of
7-nAChRs (Anand et al. 1993
;
Conroy and Berg 1998
; Couturier et al.
1990
; Schoepfer et al. 1990
) and their high
relative calcium permeability could, in principle, enable them to
influence a wide array of calcium-dependent events in neurons. A number
of such target events have already been demonstrated, including
transmitter release from presynaptic terminals (Aramakis and
Metherate 1998
; Coggan et al. 1997
;
Gray et al. 1996
; Guo et al. 1998
;
Li et al. 1998
; McGehee et al. 1995
; Radcliffe and Dani 1998
), regulation of
coexpressed postsynaptic receptors (Mulle et al. 1992a
),
neurite outgrowth (Chan and Quik 1993
; Fu and Liu
1997
; Fu et al. 1998
; Pugh and Berg
1994
), and second-messenger cascades (Vijayaraghavan et
al. 1995
).
In view of the calcium influx neuronal nAChRs can generate
(Mulle et al. 1992a; Rathouz and Berg
1994
; Vernino et al. 1994
), it is not
surprising that several receptor species have been shown to be
regulated by extracellular calcium. The predominant effect is a
potentiation of receptor response as a function of increasing calcium
over the low millimolar range (Booker et al. 1998
;
Eisele et al. 1993
; Fenster et al. 1997
;
Mulle et al. 1992b
; Vernino et al. 1992
).
One of the most illuminating accounts has been a study of
heterologously expressed
7-nAChR mutants, which has identified
specific domains in the N-terminal region that mediate the effects of
extracellular calcium (Galzi et al. 1996
). The responses
of native
7-nAChRs on rat hippocampal neurons also undergo
calcium-dependent potentiation (Bonfante-Cabarcas et al. 1996
). In vivo, this form of regulation may have greatest
biological relevance during periods of intense synaptic activity when
local decreases in extracellular calcium (Benninger et al.
1980
; Heinemann et al. 1990
; Livsey et
al. 1990
; Pumain and Heinemann 1985
) could decrease receptor function and provide a type of negative feedback control (Amador and Dani 1995
).
One of the richest sources of 7-nAChRs is the chick ciliary
ganglion, which contains ~106 such receptors per neuron
at the end of development (Chiappinelli and Giacobini
1978
; Corriveau and Berg 1994
). The ganglion has two classes of neurons in about equal numbers: large ciliary neurons that innervate striated muscle in the iris and ciliary body, and small
choroid neurons that innervate smooth muscle in the choroid layer
(Landmesser and Pilar 1974
). The ciliary neurons, which receive innervation from midbrain neurons via large presynaptic calyces, are capable of sustaining synaptic transmission at rates in
excess of 100 Hz at maturation (Dryer 1994
). Although
7-nAChRs appear to be excluded from postsynaptic densities on
ciliary neurons (Jacob and Berg 1983
; Loring et
al. 1985
; Wilson Horch and Sargent 1995
), they
nonetheless are capable of generating large synaptic currents
(Ullian et al. 1997
; Zhang et al. 1996
).
Recent evidence indicates that the receptors are concentrated on
somatic spines that are tightly folded into discrete clusters on the
neuron surface (Shoop et al. 1999
). A much less abundant
class of receptors containing the
3,
4,
5, and sometimes
2
gene products (
3*-nAChRs) are concentrated partly in postsynaptic
densities on the neurons (Conroy and Berg 1995
;
Jacob et al. 1984
; Loring and Zigmond
1987
; Vernallis et al. 1993
; Wilson Horch
and Sargent 1995
) and also contribute to the synaptic response
(Chiappinelli 1983
; Loring et al. 1984
; Ullian et al. 1997
; Zhang et al. 1996
).
The present studies were undertaken for three reasons. First, it was
important to determine whether conclusions reached about calcium-dependent potentiation of heterologously expressed 7-nAChRs (Galzi et al. 1996
) could be extended to native
7-nAChRs concentrated primarily on somatic spines and to
3*-nAChRs on the same neurons. Second, the information was necessary
for understanding in subsequent studies how calcium-dependent
regulation of the receptors might explain relationships between
synaptic current amplitude and stimulation frequency at calyx synapses.
Last, any comprehensive model describing the impact of nAChR activation
on calcium accumulation in postsynaptic compartments such as somatic
spines must take into account both the availability and regulatory
influence of extracellular calcium.
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METHODS |
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Cell preparations
Dissociated ciliary ganglion neurons were prepared from
embryonic day 13 chick ciliary ganglia using a modification of methods previously described (Margiotta and Gurantz 1989). The
ganglia were dissected from the embryo, hemisected, and incubated with 1 mg/ml collagenase (type 1, Worthington Biochemical) for 20-30 min at
37°C. The dissociation medium contained (in mM) 150 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH
7.4 (with NaOH). After collagenase treatment, the ganglia were
transferred to culture medium made up of Eagle's minimal essential
medium (GIBCO) supplemented with 10% (vol/vol) heat-inactivated horse serum (Gemini) and 3% (vol/vol) embryonic eye extract (Nishi
and Berg 1981
). The cells were dispersed by trituration with a
fire-polished Pasteur pipette and plated on glass coverslips or on
35-mm plastic tissue culture dishes (Falcon) that had been coated with
poly-D-lysine (1 mg/ml). Dissociated cells were used within
6 h of plating and were kept in a humidified tissue culture
incubator with 95% air-5% CO2 at 37°C until use.
Some experiments were performed on the cell line QT6-7, a quail
fibroblast cell line that was stably transfected with a chicken
7-nAChR gene construct (Kassner and Berg 1997
). The
cell line was passaged every second or third day in DMEM high glucose
medium (Cellgro, Mediatech-Fisher) supplemented with glutamine,
penicillin, streptomycin, nonessential amino acids, and 10% fetal
bovine serum (Gemini BioProducts, Calabasas, CA). QT6-
7 cultures
were maintained in 92% air-8% CO2 in a humidified
incubator at 37°C.
Electrophysiology
Whole cell patch-clamp recordings were obtained from isolated
cells as previously described (Hamill et al. 1981;
Zhang and Berg 1995
). All experiments were carried out
at room temperature. Electrical access was achieved either
conventionally by rupturing the membrane under the patch pipette or
noninvasively by using the perforated patch method (Horn and
Marty 1988
; Rae et al. 1991
). Patch pipettes
were pulled from thin-walled (1.5 mm OD) borosilicate glass (type N51,
Drummond Scientific, Broomall, PA) using a Sutter Instruments P-87
pipette puller and had resistances of 1-1.5
for perforated patch
pipettes and 2-2.5 M
for conventional patch pipettes. For
conventional (dialyzing) whole cell experiments the intracellular
solution contained (in mM) 130 CsCl, 2 MgCl2, 10 Cs4BAPTA, 4 Mg-ATP, and 10 HEPES, pH 7.2 (with
CsOH). The intracellular solution in perforated-patch experiments
contained (in mM) 145 CsCl, 2 MgCl2, and 10 HEPES, pH 7.2 (with CsOH). To prepare the pipettes for perforated
patch-clamp recording, stock solutions of amphotericin B were prepared
as previously described (Rae et al. 1991
). Intracellular
solution containing 400 µg/ml amphotericin B was then used to
backfill the pipette while intracellular solution alone was used to
fill the tip. Cells were discarded unless the seal formation permitted
low resistance access within 10-20 min (series resistance
10 M
)
due to the amphotericin B. Series resistance for both recording
configurations was in the range of 5-10 M
; series resistance
compensation of 80% was applied.
The external solution for whole cell recordings was (in mM) 150 NaCl,
2.5 KCl, 2 CaCl2, 1 MgCl2,
10 glucose, and 10 HEPES, pH. 7.4 (with NaOH). In some experiments, the
2 mM CaCl2 was replaced by either 0.1 mM EGTA, 2 mM MgCl2, 2 mM BaCl2, or 2 mM SrCl2. Nicotine and ACh were applied via a
rapid application system as previously reported (Zhang et al.
1994). Briefly, control and agonist-containing solutions were
delivered onto the cells from a linear array of fused glass tubes (0.35 mm ID and 0.45 mm OD; Polymicro Technologies, Phoenix, AZ). Flow of
solution through the individual tubes was induced by gravity feed and
regulated by solenoid valves (General Valve, Fairfield, NJ). Movement
of the tube array was mediated by a piezoelectric bender actuator (P-803.40, Polytec PI) connected to a voltage generator (Burleigh). The
rate of solution exchange at the cell surface was estimated by
monitoring the liquid junction potential change at an open patch
pipette and was found to require <5 ms. Agonists were tested on cells
at 1-min intervals to allow recovery from desensitization.
Perforated patch-clamp recordings in the absence of extracellular calcium could only be maintained for short periods of time. Accordingly, to test agonist responses in the absence of calcium, the rapid application was first used to perfuse the cell with solution containing 2 mM calcium (control condition), then very briefly (~10 ms) in solution containing 0.1 mM EGTA instead of calcium, and then in the same calcium-free solution containing the agonist for the 1-s test period before returning to the control condition. This minimized mixing of the calcium-containing solution and the agonist-containing calcium-free solution. Cells could be cycled several times through such a protocol before showing significant rundown, i.e., incomplete recovery on return to the control condition. Conventional patch-clamp recording in calcium-free medium appeared to be much more stable, although again the nicotinic response showed some rundown or incomplete recovery over extended periods.
Membrane currents were amplified and filtered at 1 kHz using an
Axopatch 200A patch clamp amplifier (Axon Instruments, Foster City,
CA), digitizing with DigiData 1200B (Axon Instruments), and acquired at
2-5 kHz using Clampex6 (Axon Instruments) on a personal computer. Peak
amplitudes and kinetics of the currents were analyzed using Clampfit 6 (Axon Instruments). The decay phase of the whole cell nicotinic
responses was usually fit with two exponentials to calculate the values associated with a rapidly decaying
7-nAChR component and a
more slowly decaying
3*-nAChR component that contained some
7-nAChR contribution as well (Zhang et al. 1994
) (and
see RESULTS). Unless otherwise indicated, values are presented as means ± SE and were evaluated for significance using either the paired or unpaired t-test as appropriate.
When cells were to be treated with -bungarotoxin (
Bgt), the toxin
was applied at 100 nM at 37°C and either tested throughout (for
"before and after" comparisons on the same cell) or tested after
1-2 h (for analyzing populations of cells); 20 nM
Bgt was also
included in the recording solution. In some experiments 0.5 µM
tetrodotoxin was added to the bath, but usually it was omitted because
previous experiments showed it to be unnecessary when recording
nicotinic responses in the neurons with patch-clamp techniques
(Zhang et al. 1994
).
Materials
White Leghorn chick embryos were obtained locally and maintained
at 37°C in a humidified incubator. Bgt was purchased from Biotoxins (St. Cloud, FL). All other reagents were purchased from Sigma
unless otherwise indicated.
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RESULTS |
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Calcium potentiation of native 3*-nAChR responses
The dependence of neuronal nAChR responses on extracellular
calcium was examined in dissociated chick ciliary ganglion neurons using whole cell patch-clamp recording. The perforated patch-clamp technique was used to avoid dialysis of intracellular components (Horn and Marty 1988; Rae et al.
1991
). Initial experiments targeted the
3*-nAChR response
because it could be isolated pharmacologically with
Bgt
(Ullian et al. 1997
; Zhang et al. 1994
,
1996
). In the presence of 2 mM extracellular calcium,
rapid application of 20 µM nicotine to embryonic neurons voltage
clamped at
60 mV elicited a large biphasic inward current (Fig.
1A). Application of 100 nM
Bgt to the same neurons indicated that the toxin produced a dramatic
decline in the peak and a smaller decline in the sustained portion of
the whole cell response. The
Bgt-sensitive and -resistant portions
are produced by
7-nAChRs and
3*-nAChRs, respectively (Ullian et al. 1997
; Zhang et al. 1994
,
1996
). Thus the
7-nAChR response included a large
rapidly desensitizing component and a smaller slowly desensitizing
component, whereas the
3*-nAChR response was predominantly slow in
desensitization as previously inferred from studies on cell populations
(Zhang et al. 1994
). The
3*-nAChR response also had a
slower initial rise time than the
7-nAChR response, causing the peak
current to be slightly delayed with respect to the peak obtained from
the same neurons before toxin treatment (Fig. 1A). The
mean amplitude of the
3*-nAChR peak response seen in the presence of
Bgt was about a quarter of the original whole cell peak response
(Fig. 1B).
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The calcium dependence of the 3*-nAChR response was examined by
first blocking
7-nAChRs with
Bgt and then comparing the remaining
whole cell responses obtained from the same cells in 0 and 2 mM
extracellular calcium. Removal of extracellular calcium or replacing it
with EGTA caused a rapid and reversible reduction in the
3*-nAChR
response (Fig. 2A and
B). No change was seen in the rise time of the response
(Fig. 2C) or in the decay constant describing the slowly
desensitizing
3*-nAChR response (Fig. 2D). About
one-third of the cells also displayed a second small component (<20%
of the peak) with an intermediate time course in 2 mM calcium; this
second component appeared to be lost when calcium was removed (Fig.
2D). Small components with an intermediate rate of decay have been seen previously in some ciliary neurons (Ullian et al. 1997
; Zhang et al. 1994
, 1996
),
but their significance is unclear. The results indicate that calcium
potentiates the response of native
3*-nAChRs expressed by chick
ciliary ganglion neurons.
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Potentiation of native 7-nAChR responses
The effects of calcium on the 7-nAChR response were assessed by
focusing on the rapidly desensitizing component that dominated the
whole cell peak response. Because the
3*-nAChR response elicited by
nicotine has a slower rise time than the
7-nAChR response, the
former actually contributes relatively little to the whole cell peak
response seen in the absence of
Bgt. Thus comparing the amplitudes
of the
Bgt-sensitive and -resistant currents in the same cells shows
that in 2 mM calcium
7-nAChRs produce 93 ± 2% (mean ± SE; n = 10 cells) of the peak response. At slightly later times, of course (e.g., at the peak of the
Bgt-resistant response), the relative contribution of
3*-nAChRs is much larger. Accordingly, the whole cell peak response in the absence of
Bgt was
taken in the experiments below to represent a good approximation of a
pure
7-nAChR response.
Removal of extracellular calcium from the perfusion solution or
replacing it with EGTA quickly produced a marked decrease in the whole
cell peak response caused by 20 µM nicotine. The effect, tested at
1-min intervals to avoid desensitization, was rapidly reversible (Fig.
3A). The extent of the
decrease was substantial (Fig. 3B) and approximated that
seen with 3*-nAChRs. The fact that calcium removal had no effect on
the rise time of the
3*-nAChR response (Fig. 2C) and the
fact that it reduced both the
3*-nAChR and whole cell peak responses
to similar extents justified using the latter to assess
7-nAChR
responses both in 0 and 2 mM calcium.
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The kinetics of the response were also changed by calcium removal (Fig.
3C). The rise time from 10 to 90% peak amplitude
increased over threefold (Fig. 3D). Because the small
proportion of the peak current produced by 3*-nAChRs should not have
been changed significantly by calcium removal, the results suggest that
the rate of activation for
7-nAChRs depends in part on the presence of extracellular calcium. Removal of extracellular calcium nominally increased the decay constant (
1) associated with
7-nAChR desensitization, but the reliability of the determination
may have been compromised by the small amplitudes of the
7-nAChR and
3*-nAChR responses under these conditions. No change was seen in the
decay constant (
2) associated with the slowly decaying
response that largely, although not exclusively, represented the
contributions of
3*-nAChRs (Fig. 3E).
The potentiating effect of calcium on the whole cell peak response increased smoothly with calcium concentration up to a peak at 2 mM; a diminished response was seen at 4 mM calcium (Fig. 4). Other divalent ions were able to substitute for calcium in producing the increased nicotinic response. Most effective was strontium (Fig. 5). Barium was slightly less effective than calcium, whereas magnesium was ineffective in potentiating the response.
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Mechanism of calcium potentiation
Extracellular calcium has been shown to potentiate nAChR responses
both by increasing the affinity for agonist (i.e., shifting the
dose-response curve) and by increasing the probability of channel
opening (Bonfante-Cabarcas et al. 1996; Booker et
al. 1998
; Eisele et al. 1993
;
Fenster et al. 1997
; Galzi et al. 1996
; Mulle et al. 1992b
; Vernino et al. 1992
).
A shift in the dose-response does contribute to the calcium
potentiation of ciliary ganglion nAChR responses, but it is a small
effect. This can be seen by comparing the calcium dependence of
responses elicited by 20 and 100 µM nicotine. Removing calcium or
replacing it with EGTA reduced the peak response elicited by 100 µM
nicotine to a residual 42.1 ± 3.3% (n = 9 cells) of that seen in 2 mM calcium. Performing the same experiments
with 20 µM nicotine as agonist yielded a residual 31.2 ± 1.1%
(n = 9) instead. The different extents of reduction
obtained with 20 and 100 µM nicotine is small but statistically significant (P < 0.003). Part of the
calcium-dependent potentiation, then, appears to result from a shift in
the dose-response curve, which makes a subsaturating test concentration
(e.g., 20 µM nicotine) more effective. This is unlikely to account
for the entire effect, however, because calcium also enhances the
amplitude of responses elicited by 100 µM nicotine, which represents
a concentration at the top of the dose-response curve (Zhang et
al. 1994
).
Another potential explanation for the calcium dependence of the
nicotinic response, particularly that of 7-nAChRs, is that calcium
ions may be needed to carry the current, given the high relative
calcium permeability of such receptors (Bertrand et al. 1993
; Seguela et al. 1993
). An inspection of the
current-voltage relationship for ciliary ganglion
7-nAChRs, however,
shows that most of the current is actually carried by monovalent
cations under normal physiological conditions. The responses show
strong rectification, as is true of all neuronal nAChRs tested to date (McGehee and Role 1995
; Sargent 1993
),
but reverse near 0 mV (Fig. 6). Most
important, the rapidly desensitizing
7-nAChR response is at least as
much dependent on extracellular calcium at +60 mV as it is at
60 mV
(compare the responses at 2 vs. 0 mM calcium for a given voltage; Fig.
6A). In no case should removal of extracellular calcium
decrease the response if calcium serves only to carry the current and
the current is outward, as seen at +60 mV. Additional evidence can be
obtained by replacing extracellular sodium either with isoosmotic
sucrose or with the impermeant ion
N-methyl-D-glucamine. Complete substitution
of sodium with sucrose reduced the peak whole cell nicotinic response
to a residual 1.1 ± 0.1% (n = 6) of control
values while exchanging only half of the sodium reduced the peak to a
residual 37.1 ± 1.3% (n = 6). The reductions
are consistent with sodium carrying most of the charge entering the cell under physiological conditions, even for
7-nAChRs at the peak
of the response.
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The site of calcium action in potentiating the 7-nAChR response is
not intracellular. This can be shown by using conventional patch-clamp
recording to dialyze the interior of the cell with the calcium chelator
1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). The mean peak response elicited by 20 µM nicotine in
the presence of 2 mM calcium was 3.60 ± 0.36 nA
(n = 7) when recorded with conventional patch
pipettes containing 10 mM BAPTA (allowing 3-5 min for intracellular
dialysis before recording). This is not significantly different from
the 3.49 ± 0.24 nA (n = 9) obtained with
perforated patch-clamp recording (P > 0.5). In
addition, intracellular dialysis with 10 mM BAPTA via the patch pipette
did not significantly alter the dependence of the
7-nAChR response
on extracellular calcium. The decrement in peak response accompanying
calcium removal was rapid, reversible, and comparable in magnitude
(Fig. 7) to that seen above with
perforated patch-clamp recording.
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Potentiation of 7-nAChR responses in nonneuronal cells
The experiments with ciliary ganglion 7-nAChRs made use of
nicotine as an agonist because it permitted the best temporal resolution of
7- and
3*-nAChR peak responses. The availability of
a stably transfected cell line expressing a chicken
7 gene construct
(Kassner and Berg 1997
) offered an opportunity to test the calcium potentiation of
7-nAChRs with the natural agonist ACh
because no other confounding nAChRs are present on the cells. ACh was
also the agonist of choice because it did not desensitize the receptors
so rapidly and thereby permitted concentrations to be used that
elicited larger responses than possible with nicotine under the
circumstances. This latter feature was essential because of the small
7-nAChR responses usually encountered in the transfected cells.
Rapid application of 1 mM ACh to the stable transfectants induced a
current that was reversibly reduced by removal of extracellular calcium
or replacement with EGTA (Fig.
8A). Considerable variation was seen in the amplitude of the peak response, which ranged from 50 to
550 pA among cells voltage clamped at 60 mV. Most responses fell in
the 100- to 200-pA range. The extent of the reduction caused by calcium
removal (Fig. 8B) was comparable with that calculated for
native
7-nAChRs on ciliary ganglion neurons coexpressed with
3*-nAChRs. The rise time showed a small but significant increase following calcium removal (Fig. 8C) in qualitative agreement
with the increase inferred above for native
7-nAChRs. Calcium
removal did not change the decay constant of the response (Fig.
8D). The results demonstrated that calcium potentiation
applies both to nicotine and ACh activation of the receptors and that
the potentiation seen with ciliary ganglion
7-nAChRs is a function
of the receptor itself, consistent with previous reports on
heterologously expressed receptors in Xenopus oocytes
(Galzi et al. 1996
); it is not conferred by
neuron-specific modification or auxiliary components.
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DISCUSSION |
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The principal results reported here are that calcium potentiates
the responses of both the native 7-nAChR and
3*-nAChRs on chick
ciliary ganglion neurons, and that it does so at extracellular sites.
The effects are most pronounced at physiological concentrations of
extracellular calcium and are rapidly reversible. Part of the potentiation at subsaturating agonist concentrations may involve an
increase in agonist affinity but much of the potentiation, by
inference, must also involve increased channel activity underlying the
response. As pointed out for other systems (Amador and Dani 1995
), dependence on extracellular calcium may offer a
regulatory mechanism for coupling neural activity via local calcium
depletion to synaptic currents generated by neuronal nAChRs. This could be particularly important for
7-nAChRs, which have the capacity to
elevate intracellular calcium levels substantially (Bertrand et
al. 1993
; Seguela et al. 1993
;
Vijayaraghavan et al. 1992
).
Calcium influx does not play a role in the potentiation of ciliary
ganglion nAChRs responses. This follows from the current-voltage studies and from the ion substitution experiments. Further, the experiments with BAPTA intracellular dialysis demonstrated that intracellular calcium was unnecessary for the potentiation. The calcium
concentration producing the maximal effect was close to that expected
for normal extracellular calcium levels, and other divalent cations
such as barium and strontium (but not magnesium) readily substituted
for calcium as seen for other neuronal nAChRs (Booker et al.
1998).
Previous studies have demonstrated that a variety of neuronal nAChR
subtypes can be modulated by calcium, and that the mechanisms include
increases in agonist affinity, increases in single-channel open
probability, and possibly increases in agonist-induced receptor desensitization (Amador and Dani 1995; Booker et
al. 1998
; Eisele et al. 1993
; Fenster et
al. 1997
; Mulle et al. 1992b
; Vernino et
al. 1992
). Directly relevant here are studies on heterologously expressed chicken mutant and wildtype
7-nAChRs, showing that extracellular binding sites for calcium in the N-terminal domain of the
receptors increase responsiveness; the potentiation was largely
consistent with an allosteric effect favoring the open channel state
(Galzi et al. 1996
). The calcium potentiation of ciliary
ganglion nAChRs described here may also involve primarily an allosteric
effect favoring the open channel state; certainly the N-terminal
extracellular sites identified in the heterologous expression studies
are likely to mediate much of the effect seen with native
7-nAChRs.
Calcium removal had no effect on the rise time of 3*-nAChR responses
but did increase the rise time of
7-nAChR responses both in ciliary
ganglion neurons and in the stably transfected fibroblast cell line.
The mechanism could involve a calcium-dependent increase in the rate of
agonist binding. If the effect were more pronounced with nicotine
binding to
7-nAChRs than with ACh binding to the receptors, it could
explain why a much more substantial increment in rise time resulted
from calcium removal in the neuronal tests than in the cell line tests.
Most 7-nAChR species examined to date have a very rapid rate of
desensitization (Alkondon and Albuquerque 1993
;
Zhang et al. 1994
; Zorumski et al. 1992
;
but see Cuevas and Berg 1998
). Acute changes in
extracellular calcium did not alter either the slowly desensitizing
rate of
3*-nAChR responses in ciliary ganglion neurons, nor did it
alter the rapidly desensitizing rate of
7-nAChR responses in the
stably transfected fibroblast cell line. A nominal calcium-dependent
increase in the rate of desensitization was seen for the rapidly
decaying component of the
7-nAChR responses in ciliary ganglion
neurons. It is unclear whether the increase is genuine and represents a
neuron-specific phenomenon (because it was not seen in the transfected
fibroblast cell line) or simply results from the limitations of using
curve fitting to resolve the
7- and
3*-nAChR components when the
combined whole cell response is small. The desensitization considered
here should not be confused with calcium-dependent rundown of
7-nAChR responses, which has been seen in rat hippocampal neurons
and occurs over a different time course (Bonfante-Cabarcas et
al. 1996
).
Comparing the responses of the same cells before and after Bgt
application makes it clear that ciliary ganglion neurons also generate
a small, slowly desensitizing
Bgt-sensitive response when activated
with 20 µM nicotine. The response overlays the
3*-nAChR response,
consistent with previous results obtained from cell populations
(Zhang et al. 1994
). The apparent lack of a slowly
desensitizing
Bgt-sensitive response in the transfected cells may
mean that it was too small to resolve or that the ACh necessitated in
those experiments as agonist had a different effect on
7-nAChR
desensitization than did nicotine. Alternatively, the slowly
desensitizing
Bgt-sensitive response may be produced by nonhomomeric
7-nAChRs or
7-nAChRs modified in a neuronally specific manner
(Cuevas and Berg 1998
; Yu and Role
1998
) or by a small population of
Bgt-sensitive
receptors lacking
7 subunits (Pugh et al. 1995
).
On chick ciliary ganglion neurons, the 7-nAChRs are highly
concentrated on somatic spines that are tightly folded into discrete clumps packed down on the cell surface (Shoop et al.
1999
). The spines are closely overlaid with calyx membrane
making extracellular space in the vicinity of
7-nAChRs extremely
limited. Because the calyx synapse is capable of high-frequency
transmission (Dryer 1994
) and because
7-nAChRs have a
high relative permeability to calcium (Bertrand et al.
1993
; Seguela et al. 1993
), it is reasonable to
suppose that synaptic activity could locally deplete extracellular
calcium levels as previously suggested for other systems (Amador
and Dani 1995
). The calcium dependence described above would
then limit subsequent
7-nAChR function. The physiological benefits
could be several. First,
7-nAChR signaling and accompanying intraspinal calcium buildup would be curtailed. Second, further depletion of extracellular calcium, which might have negative consequences for other calcium-dependent processes in the ganglion, would be avoided. Third, possible calcium efflux from spines through
7-nAChR activation as a result of a reversed calcium gradient would
be minimized. Similar considerations may also apply to
3*-nAChRs because they display the same calcium-dependent potentiation and are
also found on somatic spines (Shoop et al. 1999
).
Having identified a form of calcium-dependent regulation for nAChR function, it will now be important to determine how the regulation influences receptor effects on intracellular calcium levels. Conceivably, receptor-dependent calcium accumulation in spines provides a means for integrating information about synaptic activity. As such it may exert long-term regulatory consequences either for the synapse or for the circuit in which the ciliary neuron resides.
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ACKNOWLEDGMENTS |
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We thank Dr. Javier Cuevas of the University of South Florida Medical School for comments on the manuscript. Q.-S. Liu is an American Heart Association Postdoctoral Fellow.
This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-12601 and NS-35469 and by the Tobacco-Related Diseases Research Program.
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FOOTNOTES |
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Address for reprint requests: D. K. Berg, Dept. of Biology, 0357, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 March 1999; accepted in final form 30 April 1999.
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REFERENCES |
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