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The Journal of Neuroscience, December 15, 1998, 18(24):10335-10344
Mammalian Nicotinic Receptors with  7 Subunits That Slowly
Desensitize and Rapidly Recover from -Bungarotoxin Blockade
Javier
Cuevas and
Darwin K.
Berg
Department of Biology, University of California, San Diego, La
Jolla, California 92093-0357
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ABSTRACT |
One of the most abundant nicotinic receptors in the nervous system
is a species that contains the  7 gene product, rapidly desensitizes,
and binds -bungarotoxin with great affinity. The receptor has a high
relative permeability to calcium and performs a variety of functions
including presynaptic modulation of transmitter release and
postsynaptic generation of synaptic currents. Fast excitatory
transmission in mammalian intracardiac ganglia is mediated primarily by
nicotinic receptors, and although intracardiac ganglion neurons express
the 7 gene, no toxin-sensitive response has been detected previously
in them. We report here that whole-cell patch-clamp recordings from
freshly dissociated intracardiac ganglion neurons reveal a nicotinic
response that desensitizes slowly and is blocked by -bungarotoxin in
a rapidly reversible manner. The only rat gene previously thought
capable of forming such receptors was 9, but no evidence suggests
that the 9 gene is expressed in neurons. We find that reverse
transcription (RT)-PCR detects 7 but not 9 mRNA in the
ganglia. In addition, the pharmacology of the nicotinic response is
typical of 7-containing receptors but differs in several respects
from that expected for 9. Binding experiments with immunotethered
receptors identifies a ganglionic species that contains the 7 gene
product. Moreover, intracellular perfusion of the cells with an
anti-7 monoclonal antibody specifically reduces the amplitude of the
toxin-sensitive response. The results indicate that 7-containing
receptors are responsible for the slowly desensitizing,
toxin-reversible response and suggest that the receptors are modified
in cell-specific ways to influence their functional properties.
Key words:
nicotinic; receptors; acetylcholine; intracardiac
ganglion; neuronal; 7; -bungarotoxin; patch clamp
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INTRODUCTION |
The nicotinic acetylcholine receptor
(AChR) 7 gene is widely expressed in the nervous system and accounts
for the most abundant species of neuronal AChR in both chick and rat
(Marks et al., 1986 ; Couturier et al., 1990; Schoepfer et al., 1990;
Anand et al., 1993b; Chen and Patrick, 1997; Conroy and Berg, 1998).
When heterologously expressed in Xenopus oocytes, 7
protein assembles into homopentameric ligand-gated ion channels that
are cation-selective, rapidly desensitize, and bind -bungarotoxin
(Bgt) with high affinity (Couturier et al., 1990; Bertrand et al.,
1993; Seguela et al., 1993). Responses from native AChRs containing the
7 gene product (7-AChRs) have been reported in several systems
and have always been found to be similar to those of the homopentamer
in oocytes; namely, they rapidly desensitize and are blocked by Bgt in a long-lasting manner (Zorumski et al., 1992; Alkondon and Albuquerque, 1993; Zhang et al., 1994).
Native 7-AChRs are likely to serve a number of physiological roles.
Recent evidence indicates they can act presynaptically to modulate
neurotransmitter release (McGehee et al., 1995; Gray et al., 1996;
Coggan et al., 1997) and can function at extra- or perisynaptic
sites on neurons to generate synaptic currents as well (Zhang et al.,
1996; Ullian et al., 1997). Genetic studies have linked the receptors
to a form of schizophrenia (Freedman et al., 1997). Cell culture
analysis has suggested the receptors may be important for early
developmental events because they can be found on growing neurites
(Pugh and Berg, 1994; Fu and Liu, 1997). This diversity of function
raises the question of whether the properties of 7-AChRs vary with
cellular location to accommodate site-specific job requirements.
Most puzzling has been the repeated finding of Bgt binding on
neurons with no apparent Bgt-sensitive response (Duggan et al.,
1976; Carbonetto et al., 1978; Betz, 1981; Lipton et al., 1987; Sucher
et al., 1990; Zhang and Feltz, 1990; Sargent and Garrett, 1995). This
has frequently been the finding with mammalian autonomic neurons (Nurse
and O'Lague, 1975; Brown and Fumagalli, 1977; Ascher et al., 1979;
Mandelzys et al., 1995). Other than 7, the only known genes
that produce Bgt-binding receptors are the muscle 1 and either
the 9 in mammals or the 8 in chick. Neither the 1 nor the 9
genes are expressed in neurons (Elgoyhen et al., 1994; Karlin
and Akabas, 1996). Although the chick 8 is expressed in
neurons, it either coassembles with 7 subunits to produce heteromers
or self-assembles to produce 8-containing homomers (Schoepfer et
al., 1990; Anand et al., 1993a), and both are capable of
Bgt-sensitive responses when expressed in oocytes (Gerzanich et al.,
1994).
An interesting system to explore the nature of 7-AChR responses is
provided by mammalian intracardiac ganglia. The ganglia mediate
efferent parasympathetic input to the heart and are thought to exert
local regulation over cardiac function by integrating information from
efferent and afferent pathways of both parasympathetic and sympathetic
origin (Moravec and Moravec, 1987; Gagliardi et al., 1988). Extrinsic
and intrinsic innervation of the ganglia is predominantly cholinergic,
with activation of AChRs resulting in fast excitatory transmission
(Seabrook et al., 1990). Rat intracardiac ganglion neurons apparently
express multiple AChR subtypes, and the combination of subtypes
expressed varies among cells (Poth et al., 1997). Although many of the
neurons express the 7 gene (Poth et al., 1997), no Bgt-sensitive
responses have been detected previously in the cells (Selyanko and
Skok, 1992).
We have used whole-cell patch-clamp recording, together with rapid
application of agonist, to examine the nicotinic ACh responses of
dissociated rat intracardiac ganglion neurons. The neurons display a
slowly desensitizing response that is blocked by Bgt in a rapidly
reversible manner. Pharmacological analysis, reverse transcription (RT)-PCR, immunoprecipitation, and
intracellular dialysis with subunit-specific monoclonal antibodies
(mAbs) are each consistent with the conclusion that 7-AChRs produce
the response. The implication is that 7-AChRs can be modified or regulated to display different properties in different environments. If
7-AChRs in intracardiac ganglion neurons retain the feature of
having a high relative permeability to calcium, their ability to
sustain long-duration currents in this case is likely to empower them
with a major role in ganglionic signaling and regulation of cardiac function.
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MATERIALS AND METHODS |
Tissue preparation. Neurons from neonatal rat
intracardiac ganglia were isolated and maintained as described
previously (Cuevas and Adams, 1994). Briefly, to obtain intracardiac
ganglion neurons, we killed postnatal day 3 (P3)-P7 rats by
decapitation. The hearts were excised and placed in a saline solution
containing (in mM): 140 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 7.7 glucose,
and 10 histidine, pH 7.2 with NaOH. The atria were separated and
incubated in saline solution containing collagenase (1 mg/ml; Type 1A;
Worthington, Freehold, NJ) at 37°C for 60 min. After enzymatic
treatment, clusters of ganglia were dissected from the epicardial
ganglion plexus and dispersed by titration in a high glucose culture
medium (DMEM; Life Technologies, Gaithersburg, MD) with 10%
fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin.
The dissociated neurons were then plated on glass coverslips coated
with laminin, incubated at 37°C under a 95% air/5% CO2
atmosphere, and examined 36-72 hr later.
Chick ciliary ganglion neurons were dissociated from 14-15 d embryos
as described previously (Margiotta and Gurantz, 1989). Briefly, the
ganglia were dissected from the embryo, incubated with 1 mg/ml
collagenase for 30 min at 37°C, and transferred to culture medium
made up of Eagle's Minimal Essential Medium (Life Technologies)
supplemented with 3% (v/v) embryonic eye extract (Nishi and Berg,
1981). The cells were dispersed by trituration, plated on a substratum
of poly-D-lysine in 35 mm Costar culture dishes, and
examined 1-3 hr later.
Electrophysiological recordings. Neurons plated on glass
coverslips were transferred to a recording chamber (volume, 0.5 ml) mounted on an inverted phase-contrast microscope (magnification, 400×)
that allowed isolated cells to be identified. Membrane currents in
intracardiac neurons were studied under voltage-clamp mode using the
whole-cell patch-clamp technique (Hamill et al., 1981). Electrical
access was achieved conventionally by rupturing the membrane under the
patch pipette or via the use of the perforated-patch method (Horn and
Marty, 1988). Patch electrodes were pulled from thin-walled [outer
diameter (o.d.), 1.5 mm] borosilicate glass (Drummond Scientific,
Broomall, PA) using a Sutter Instruments P-87 pipette puller (Novato,
CA) and had resistances of 1-1.5 M . For conventional (dialyzing)
whole-cell experiments, the intracellular solution contained (in
mM): 140 CsCl, 10 glucose, 2 EGTA, and 10 HEPES, pH 7.2 with CsOH. In some conventional whole-cell experiments, cells were
dialyzed ( 10 min) with patch pipette solutions containing subunit-specific anti-AChR mAbs; the specificities of the mAbs have
been described previously [see references in Vernallis et al. (1993);
Conroy and Berg (1995)]. The intracellular solution in
perforated-patch experiments contained (in mM): 75 K2SO4, 55 KCl, 5 MgSO4, 360 µg/ml amphoterecin B, 0.6% DMSO, and
10 HEPES, pH 7.2 with N-methyl-D-glucamine. The
procedures for achieving electrical access with amphoterecin B were
identical to those described previously (Cuevas et al., 1997) and
resulted in series resistance  3 M after compensation
(50%).
Membrane currents were amplified and filtered (5 kHz) using an Axopatch
200A (Axon Instruments, Foster City, CA) patch-clamp amplifier,
digitized with a Digidata 1200B (Axon Instruments), and acquired (20 kHz) using Clampex 6 (Axon Instruments) on a pentium/133 MHz computer.
Peak amplitude and kinetics of agonist-evoked currents were analyzed
using Clampfit 6 (Axon Instruments).
The external solution for whole-cell recordings was physiological
saline solution containing (in mM): 140 NaCl, 3 KCl,
2.5 CaCl2, 1.2 MgCl2, 7.7 glucose, and 10 HEPES, pH 7.2 with NaOH. Agonists and antagonists were
applied via a rapid application system as reported previously (Zhang et
al., 1994). Briefly, control and drug-containing solutions were
delivered onto the cell soma from a linear array of glass tubes (inner
diameter, 250 µm; o.d., 350 µm; 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 bimorph connected to a voltage generator (Burleigh,
Fishers, NY). The rate of solution change expected to be observed by
the cell was determined by recording the liquid junction potential
change from an open patch pipette and was < 5 msec.
Solid-phase immunoprecipitations. Solid-phase
immunoprecipitation assays were conducted as described previously
(Conroy and Berg, 1995). Atria from neonatal rats (4 and 14 d old)
were dissected as described above, and the medial region containing the
pulmonary veins and the superior and inferior vena cava was isolated.
These segments were homogenized in 2% (w/v) Triton X-100 extraction buffer and incubated at 4°C for 1 hr. Extracts were then centrifuged at 17,000 × g for 20 min, and the supernatant fraction
was collected. Rat brain extracts were prepared from whole brains of
neonatal rats (20 d old) using methods described previously (Conroy et al., 1992). Aliquots were incubated overnight at 4°C in microtiter wells precoated with anti-7 mAb 319 to immunotether AChRs containing the 7 gene product. Receptor binding was quantified with
125I-Bgt. Nonspecific binding was determined by
including either 1 µM unlabeled Bgt or 1 mM nicotine in the binding reaction with 125I-Bgt and was subtracted from total binding to obtain
specific binding. In some experiments the anti- 2 mAb 270 and the
anti-8 mAb 308 were used as immunotethering antibodies [for mAb
specificities, see references in Vernallis et al. (1993); Conroy and
Berg (1995)].
Epibatidine binding was determined using a filter binding assay (Conroy
and Berg, 1995). Protein extracts (25 µl aliquots) were incubated in
2 nM [3H]epibatidine for 2 hr at room
temperature. The reactions were then diluted with 4 ml of wash buffer
[0.05% (w/v) Triton X-100 in 10 mM Tris, pH 7.5], and
the solution was immediately filtered through Whatman GF/B filters
(Maidstone, UK) presoaked for 1 hr in 0.5% polyethyleneimine. Filters
were rinsed twice more with wash buffer and then counted by liquid
scintillation (Ecoscint H; National Diagnostics, Atlanta, GA).
Nonspecific binding was determined by including 1 mM
nicotine in the binding reaction with
[3H]epibatidine.
RT-PCR. The use of RT-PCR for detection of AChR gene
expression in cultured neurons from rat intracardiac ganglia has been described previously (Poth et al., 1997). Briefly, RNA was extracted (RNeasy; Qiagen, Hilden, Germany) either from a dish of cultured intracardiac neurons containing ~100 neurons plus a number of other
cell types (e.g., cardiac myocytes, Schwann cells, and fibroblasts) or
from intact intracardiac ganglia and associated tissue (same as in
culture preparations). RNA was reverse-transcribed in a 20 µl
reaction volume using a Life Technologies SuperScript Preamplification System kit. Negative controls including an RT reaction without reverse
transcriptase and a PCR reaction with only water were conducted to
eliminate the possibility of false positives because of contaminating
cDNA. Primers for 7 and 9 transcripts were identical to those
used previously [7 (Poth et al., 1997); 9 (Elgoyhen et
al., 1994)]:
7(forward)-GGAGTGAAGAATGTTCGTTTTCCAGATGG, 7(reverse)-CCCTGGCTCTGCTGGTATTCTTGC,
9(forward)-CTAATGGTGGCAGAGATCATGCCA, and
9(reverse)-TATGATCAAGACGGTCATGACAAACACCA.
They yielded product sizes of 476 and 573 bp, respectively. PCR
reactions were conducted using the Life Technologies SuperScript
Preamplification System kit, and the cycling parameters were five
cycles of 94°C for 45 sec, 55°C for 1 min, and 72°C for 1.5 min.
This was followed by 30 cycles of 94°C for 45 sec, 57°C for 1 min,
and 72°C for 1.5 min.
Restriction digestion. A restriction digestion strategy was
used to confirm the identity of PCR reaction products as being those
expected for amplification of specific cDNAs. The two 7 primer
products were gel-purified and digested with HaeII and BanII. Each of these endonucleases targeted a different exon
found in the region amplified by the primers used. Digestion of the 476 bp fragment with both enzymes produces digestion products of 321, 78, and 77 bp. Sequence analysis of cloned PCR products was performed
commercially (Retrogen, San Diego, CA).
Reagents and statistical analysis. All chemicals used were
of analytical grade. Acetylcholine chloride (ACh), cytisine chloride, atropine sulfate, nicotine, and mecamylamine chloride were purchased from Sigma (St. Louis, MO). Bgt was purchased from Biotoxins (St.
Cloud, FL) and radioiodinated using chloramine T to a specific activity
of 0.3-0.7 × 1018 cpm/mol.
[3H]Epibatidine (56.5 Ci/mmol) was a gift from
DuPont NEN (Boston, MA), and unlabeled epibatidine was purchased from
Research Biochemicals (Natick, MA). Several mAbs were generously
supplied by Dr. Jon Lindstrom (University of Pennsylvania,
Philadelphia, PA).
Data are presented as the mean ± SD unless otherwise stated and
were compared using paired or unpaired t tests as appropriate.
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RESULTS |
Bgt-sensitive ACh-evoked currents
Whole-cell patch-clamp techniques were used to record ACh-induced
currents from dissociated rat intracardiac ganglion neurons maintained
1-3 d in culture. Rapid focal application of agonist was used to
minimize loss of response because of receptor desensitization. Figure
1A shows a
representative membrane current response evoked by 500 µM
ACh from a neuron electrically accessed with the amphoterecin B
perforated-patch method and voltage clamped at  60 mV. ACh elicited a
transient inward current that desensitized during the 2 sec exposure to
agonist. After a 10 min application of 100 nM Bgt, the
ACh-evoked current decreased by >40% in amplitude (Fig.
1A). Mean values of 2.3 ± 0.3 and 1.3 ± 0.3 nA (n = 5 cells) were obtained for the peak
response in the absence and presence, respectively, of 100 nM Bgt. The Bgt-induced decrement was statistically
significant (p < 0.02). Similar values for the
peak ACh-induced currents plus and minus Bgt were observed when
cells were electrically accessed using the conventional patch-clamp
(dialyzing) whole-cell recording configuration: 1.6 ± 0.2 and
2.5 ± 0.2 nA, respectively (n = 4 cells).
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Figure 1.
Bgt blocks a slowly desensitizing ACh-evoked
current in rat intracardiac neurons. A,
B, Whole-cell currents evoked by rapid focal application
of 500 µM ACh to the soma of an isolated rat intracardiac
ganglion neuron (A) and an isolated chick ciliary
ganglion neuron (B), each voltage clamped at 60
mV, in the absence (Control) and presence of 100 nM Bgt. C, Net Bgt-sensitive
ACh-evoked current, determined by subtracting the current induced by
ACh in the presence of 100 nM Bgt from that recorded in
the absence of the toxin for the experiments shown in A
and B.
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For comparison, ACh-induced currents were also recorded from
dissociated chick ciliary ganglion neurons before and after application of 100 nM Bgt (Fig. 1B). As with
intracardiac neurons, the cells were electrically accessed with the
perforated-patch method and were voltage clamped at 60 mV. The time
course of the Bgt-sensitive current in both cell types was
determined by subtracting the ACh-evoked current recorded in the
presence of Bgt from that recorded in its absence in the same cell
(Fig. 1C). The Bgt-sensitive response in rat intracardiac
neurons decays much more slowly than does the response in ciliary
ganglion neurons. The decay phase of the Bgt-sensitive response in
intracardiac neurons was best fit by the sum of two exponential
functions with decay half-times of 170 ± 20 and 930 ± 70 msec (n = 4 cells).
Concentration dependence and reversibility of Bgt blockade
The concentration dependence of the Bgt blockade was examined
by comparing the peak amplitude of the ACh-induced current before and
after application of Bgt at a range of concentrations (10 pM to 1 µM). Figure
2A shows a set of
currents evoked in this manner from a single neuron voltage clamped at
60 mV. Approximately 80% of the neurons (32 out of 41 cells)
revealed an Bgt-sensitive ACh response. The peak amplitude of
ACh-evoked currents in the presence of Bgt was normalized to the
maximum response from the same neuron in the absence of Bgt, and the
results from a number of cells were compiled to generate a plot of mean
peak amplitude versus Bgt concentration (Fig. 2B).
A fit of the data using a single-site adsorption isotherm indicates
half-maximal inhibition (IC50) at 120 pM
and a maximal inhibition of 47 ± 2% at 1 µM Bgt (n = 7).
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Figure 2.
Dose-dependent inhibition of nicotinic ACh-evoked
currents by Bgt. A, Whole-cell currents evoked from a
single neuron by focal application of 500 µM ACh in the
absence (Control) and presence of Bgt (1 and
100 nM). The holding potential was 60 mV.
B, ACh-evoked whole-cell current amplitude at 60 mV
normalized to values obtained from the same cells in the absence of
Bgt and plotted as a function of Bgt concentration. Data points
represent mean ± SEM (n = 7-12 determinants;
25 neurons). The curve represents a best fit to the data using a
single-site adsorption isotherm with half-maximal inhibition at 120 pM Bgt and a maximal inhibition of 47%.
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The duration of Bgt blockade was determined by comparing the peak
amplitude of ACh-evoked currents in the same neuron before, during, and
after exposure to 100 nM Bgt. Results from individual intracardiac ganglion neurons suggest the response largely recovers after only a few minutes of rinsing to remove toxin (Fig.
3A). For comparison, similar
experiments were performed on chick ciliary ganglion neurons, which
have been reported previously to show no reversibility of the Bgt
blockade over short times (Zhang et al., 1994). A 10 min wash under the
same conditions used for the intracardiac neurons allows no recovery of
the Bgt-sensitive component in ciliary ganglion neurons (Fig.
3B) The peak amplitudes of the ACh-evoked currents
obtained from individual intracardiac ganglion neurons throughout the
procedure were normalized to the maximum response obtained from the
same neuron before application of Bgt application. Compiling such
results from a number of neurons indicates that the onset of blockade
in 100 nM Bgt occurs within 2 min but quickly reverses
(Fig. 3C). After 10 min of rinsing to remove the
toxin, the ACh-evoked currents had recovered to 87 ± 5% of
control values (n = 6).
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Figure 3.
Reversibility of Bgt-induced blockade.
A, B, A family of currents evoked by 500 µM ACh recorded from a single rat intracardiac ganglion
neuron (A) and a single embryonic day 14 chick
ciliary ganglion neuron (B) at 60 mV in the
absence (Control) and presence of 100 nM Bgt and after washout of toxin for the indicated time
periods. C, ACh-evoked whole-cell current amplitudes
before application (Control), during application
(Bgt), and after removal (Wash) of 100 nM Bgt. Values have been normalized to the maximal
response from the same cell and plotted as a function of time.
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AChR gene transcripts: 7 versus 9
The quick reversibility of the Bgt blockade and the slow
desensitization of the Bgt-sensitive response in rat intracardiac ganglion were unexpected. Although many of the neurons express the 7
gene (Poth et al., 1997), 7-AChRs in other systems generate rapidly
desensitizing currents that are essentially irreversibly blocked by
Bgt (Zorumski et al., 1992; Alkondon and Albuquerque, 1993; Zhang et
al., 1994; Blumenthal et al., 1997). The only candidates for
AChRs in rat that produce slowly desensitizing currents that are
reversibly blocked by Bgt are those composed of the 9 gene product (Elgoyhen et al., 1994). The 9 gene appears to be expressed exclusively in non-neuronal cells (Elgoyhen et al., 1994), but it
seemed prudent to test whether 9 transcripts could be detected in
intracardiac ganglia. This was done using RT-PCR.
Total RNA was extracted from cultures of rat intracardiac neurons,
reverse-transcribed, and amplified by PCR for 7 and 9 transcripts
(Fig. 4A). The primers
were designed to span introns so that false positives resulting from
genomic DNA contamination could be distinguished. Reactions with 7
primers generated a band having the expected size for the 7 product
(476 bp) from intracardiac ganglion RNA. No band of the size expected
for the 9 product (573 bp) was generated from intracardiac ganglion
RNA when 9 primers were used. Positive controls for the RT-PCR were performed with RNA extracted from neonatal rat pituitary ganglia because both 7 and 9 mRNAs are known to be present in this case (Elgoyhen et al., 1994). Both 7 and 9 products were generated using the appropriate primers (Fig. 4A). Two sets of
negative controls were conducted; these include omission of reverse
transcriptase from the RT incubation and substitution of water for
template in the PCR incubation (Fig. 4A). Restriction
digestion (Lambolez et al., 1992) provided additional evidence that the
major PCR product obtained with the 7 primers (~476 bp) originated
from the 7 transcript (Fig. 4B). The digestion
yielded products of the predicted sizes (321, 77, and 78 bp). Sequence
analysis confirmed that the major PCR product had the expected 7
sequence; the smaller product of ~390 bp (Fig. 4A)
was not 7 in origin (data not shown).
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Figure 4.
RT-PCR analysis of 7 and 9 gene expression
in rat intracardiac ganglion neurons. A, RT-PCR
products. Primers/template RNA: lane 1 (on the
left), 100 bp standards; lane 2,
7/intracardiac ganglion; lane 3, 9/intracardiac
ganglion; lane 4, 7/rat pituitary; lane
5, 9/rat pituitary; lane 6, 7 + 9/water; lane 7, 7 + 9/rat intracardiac without
RT. The expected product sizes are 476 bp for 7 and 573 bp for 9.
B, Restriction digest of the RT-PCR product amplified
from intracardiac ganglion RNA with 7 primers. Lane 1
(on the left), 100 bp standards; lane 2,
HaeII and BanII digest. Expected product
sizes are 321, 78, and 77 bp for 7 transcripts. Neg.,
Negative.
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Pharmacology of Bgt-sensitive currents
The pharmacology of Bgt-sensitive ACh responses in dissociated
intracardiac ganglion neurons was examined to determine whether the
currents had the properties expected for 7-AChRs. Although ACh
activates both 7- and 9-AChRs, cytisine and nicotine activate only the former. Cytisine fails to activate 9-AChRs, whereas nicotine blocks such receptors (Elgoyhen et al., 1994). The M1 muscarinic agonist oxotremorine M (Oxo-M) activates both classes of
receptors, whereas the glycinergic antagonist strychnine and the
nicotinic antagonist D-tubocurarine block both.
Figure 5A shows representative
membrane currents evoked by focal application of 500 µM
nicotine, 500 µM cytisine, and 300 µM Oxo-M
in the presence and absence of 100 nM Bgt. With nicotine and cytisine as agonists, Bgt blocked 37 ± 3%
(n = 3) and 41 ± 3 (n = 3) of the
current, respectively. These values are similar to those obtained with
ACh as agonist and are consistent with the Bgt-sensitive response
being the product of 7-AChRs. With Oxo-M as agonist, Bgt blocked
87 ± 5% (n = 2) of the current. Strychnine
blocked 43 ± 6% (n = 3) of the ACh-evoked
current, a value comparable with the fractional response blocked by
Bgt (Fig. 5B). The broad spectrum nicotinic antagonist
D-tubocurarine at 100 µM blocked the
ACh-evoked responses as well as those induced by Oxo-M (Fig.
5B). The current-voltage relationship of the
Bgt-sensitive currents induced by Oxo-M is that expected for
cation-selective neuronal AChRs, namely, a linear relationship at
negative potentials, a reversal potential near 0 mV, and marked inward
rectification visible at positive membrane potentials (Fig.
6). The results support the conclusion
that the slowly desensitizing, Bgt-sensitive currents obtained from
intracardiac ganglion neurons are produced by activation of
7-AChRs.
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Figure 5.
Pharmacology of Bgt-sensitive currents in
intracardiac ganglion neurons. A, Currents evoked by
rapid focal application of 500 µM nicotine
(left), 500 µM cytisine
(middle), and 300 µM Oxo-M
(right) in the absence and presence of Bgt.
B, ACh-evoked (left and
middle) and Oxo-M-evoked (right)
responses before and after treatment with 100 nM strychnine
or 100 µM D-tubocurarine
(D-TC).
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Figure 6.
Voltage dependence of peak Oxo-M-evoked current
amplitude. A, A family of currents evoked by 300 µM Oxo-M in the presence of 100 nM atropine,
recorded from a neuron held at the indicated membrane potentials.
B, Current-voltage relationship for currents induced by
300 µM Oxo-M. Points represent mean ± SEM for three
neurons each.
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Quantification of 7-AChRs in rat intracardiac
ganglion extracts
The number of 7-AChRs present in intracardiac ganglia was
measured with a solid-phase immunoprecipitation assay. Extracts were
prepared from P4 rat intracardiac ganglia and incubated with the
anti-7 mAb 319 to immunotether 7-AChRs. Bound receptors were
quantified with 125I-Bgt. A single round yielded nearly
5 fmol of binding sites per heart equivalent of intracardiac ganglia
(Fig. 7A). A second round
yielded an additional 0.5 fmol, indicating that most of the 7-AChRs
had been collected in the first pass. The recovered extracts were then
incubated with [3H]epibatidine and filtered to
collect and quantify other classes of neuronal AChRs (Gerzanich et al.,
1995; Houghtling et al., 1995; Conroy and Berg, 1998). This procedure
yielded ~2-3 fmol per P4 heart equivalent of intracardiac ganglia
(Fig. 7A). No toxin binding was detected in the solid-phase
assay when either the anti-8 mAb 308 or the anti-2 mAb 270 was
used to immunotether AChRs, nor was any
[3H]epibatidine binding detected in the assay when
the anti-7 mAb 319 was used to immunotether AChRs (data not
shown).
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Figure 7.
Quantification of 7-AChRs using a solid-phase
immunoprecipitation assay. A, Solubilized intracardiac
ganglion 7-AChRs were immunotethered with the anti-7 mAb 319 in
two sequential rounds (Rounds 1 and 2)
and then quantified with 125I-Bgt; the recovered
supernatant fractions were then assayed for
[3H]epibatidine-binding receptors using a
polyethyleneimine filter assay (Round 3).
Bars represent the mean (± SEM) amount of binding per
single heart equivalent of intracardiac ganglia for 11 (black
bar), 8 (white bar), or 3 (shaded
bar) determinations. B, 125I-Bgt
binding sites in solubilized tissue samples (Rounds 1
and 2 summed) are shown. Bars represent
the mean (± SEM) amount of binding per single heart equivalent of
intracardiac ganglia (black bars) or an ~10-fold
excess by weight of cardiac muscle tissue (white bars),
a small portion of which might have contaminated the ganglia samples.
Eight determinations were done for P4 samples, and three determinations
were performed for P14 samples.
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The numbers of 7-AChRs per heart equivalent of ganglia did not
change dramatically during early postnatal development. Values obtained
with P14 ganglia were not significantly greater than those obtained at
P4 (Fig. 7B). The binding was specific for the ganglia
because a large excess of tissue that might have contaminated ganglia
preparations (namely, small segments of atria, ventricles, thymus
gland, aorta, and lungs) failed to display significant binding on their
own (Fig. 7B).
Selective blockade of Bgt-sensitive currents by
anti-7 mAbs
To obtain further evidence that the Bgt-sensitive currents in
intracardiac ganglion neurons are mediated by activation of 7-AChRs,
we dialyzed cells intracellularly with subunit-specific mAbs. The hope
was that mAbs specific for intracellular epitopes on particular AChR
subunits might bind to those sites on native receptors in
situ and inhibit receptor function either by occluding the channel
from the inside or by preventing allosteric or regulatory changes
required for channel opening.
Intracellular dialysis of neurons via a conventional patch pipette for
periods up to 30 min with mAb 35, which recognizes extracellular
epitopes on the 1, 3, and 5 but not 7 subunits, had no
statistically significant effect either on the total ACh-evoked current
or on that portion that could be blocked by Bgt (Fig. 8). Similarly, intracellular dialysis
with mAb 313, which is specific for an intracellular epitope on 3
subunits, had no effect on either component of the response. In
contrast, intracellular dialysis for even 10 min with mAb 319, which is
specific for an intracellular epitope on 7 subunits, produced a
significant and specific decrement in the Bgt-sensitive portion of
the ACh-evoked response (Fig. 8). Dialysis with mAb 319 for periods up
to 30 min produced an even greater selective reduction in the
toxin-blockable response. Thus only 11 ± 2% (mean ± SEM)
of the whole-cell ACh response was Bgt-sensitive at 30 min, whereas
22 ± 3% was Bgt-sensitive at 10 min (p = 0.01 for 10 vs 30 min values). Both components comprising the decay
phase of the Bgt-sensitive response were affected. No preferential
rundown of the Bgt-sensitive response occurred under these
conditions, as can be seen by comparing the 10 and 30 min values for
cells receiving the control mAbs 35 and 313 (Fig. 8). The results
provide strong evidence for the conclusion that 7-AChRs are
responsible for the slowly desensitizing ACh-evoked responses that are
reversibly blocked by Bgt.
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Figure 8.
Specific blockade of Bgt-sensitive ACh-evoked
currents by intracellular dialysis with an anti-7 mAb.
A, Whole-cell currents evoked by focal application of
500 µM ACh onto an intracardiac ganglion neuron in the
absence (Control) and presence of 50 nM Bgt after a 10 min intracellular dialysis of the cell
with either no mAb, the anti-1/3/5 mAb 35, the anti-3 mAb
313, or the anti-7 mAb 319 via the patch pipette. B,
Peak currents evoked by 500 µM ACh (white
bars) and ACh plus 50 nM Bgt (black
bars) from neurons dialyzed with the indicated mAbs. Currents
were recorded after a 10 and a 30 min dialysis. C, The
Bgt-sensitive portion of the ACh-evoked current expressed as a
percent of the peak whole-cell ACh response current for neurons after
dialysis for either 10 or 30 min with the indicated mAbs. Only the
anti-7 mAb affected the whole cell response, and it selectively
reduced the Bgt-sensitive portion of it. Bars
represent the mean ± SEM of the number of neurons indicated in
parentheses; the same number of neurons was tested in
B and C.
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DISCUSSION |
The results presented here provide the first demonstration of
ACh-evoked currents in the mammalian peripheral nervous system attributable to 7-AChRs. The currents are blocked by nanomolar concentrations of Bgt, but, unexpectedly, the blockade is rapidly reversible and the currents affected are slow to desensitize. In all
previous cases in which native 7-AChRs have been activated by rapid
application of agonist, the resulting currents were found to
desensitize rapidly and to remain inhibited long after unbound Bgt
had been removed. In addition to chick ciliary ganglion neurons (Zhang
et al., 1994), examples include rat hippocampal neurons in culture
(Zorumski et al., 1992; Alkondon and Albuquerque, 1993) and the rat
pheochromocytoma cell line PC12 (Blumenthal et al., 1997). Clearly some
populations of rat 7-AChRs are capable of rapid desensitization and
long-lasting Bgt blockade. The rapidly reversible blockade described
here may explain why no Bgt-sensitive currents have been reported
previously for mammalian autonomic neurons.
The 9 gene is known to produce AChRs that slowly desensitize and
reversibly bind Bgt, but all evidence to date indicates the gene is
not expressed in neurons (Elgoyhen et al., 1994). RT-PCR analysis in
the present experiments failed to detect 9 transcripts in rat
intracardiac ganglion RNA, although the positive controls with rat
pituitary RNA proved successful. Moreover, the pharmacology of the
Bgt-sensitive response in intracardiac ganglion neurons was
consistent with that of 7-AChRs rather than that of 9-AChRs, and
immunoprecipitation experiments confirmed the presence of 7-AChRs in
intracardiac ganglion extracts.
The number of 7-AChRs detected by 125I-Bgt binding in
P4 rat intracardiac ganglia (~5 fmol per heart equivalent) was
substantially lower than the ~30 fmol per ganglion observed for P4
rat superior cervical ganglia in the same assay (A. Roth and J. Cuevas,
unpublished results) or the ~20 fmol per ganglion for chick ciliary
ganglia at a comparable stage of development (Chiappinelli and
Giacobini, 1978; Smith et al., 1983). Intracardiac ganglia, however,
contain only ~4 × 103 neurons in aggregate
per heart equivalent, whereas rat superior cervical ganglia contain
~3 × 104 (Paxinos, 1995), and chick ciliary
ganglia contain ~3 × 103 (Landmesser and
Pilar, 1974). When the amount of 125I-Bgt binding is
normalized for the number of neurons estimated to be present, the
number of sites is comparable for the two mammalian sources (~1 fmol
per 103 neurons) but is lower than that found in
chick ciliary ganglia (~7 fmol per 103 neurons).
One of the strongest lines of evidence indicating that the
Bgt-sensitive responses arise from 7-AChRs was provided by the intracellular dialysis experiments with subunit-specific mAbs. The fact
that anti-7 mAbs selectively reduced the Bgt-sensitive ACh-evoked
current while other mAbs had no effect on either the Bgt-sensitive
or -resistant components of the response clearly implicates 7-AChRs
in producing the toxin-sensitive portion of the response. One might
have expected the anti-3 mAb 313 to reduce the Bgt-resistant
response because it almost certainly arises from 3-containing AChRs
(Poth et al., 1997). It is not known, however, whether mAb 313 recognizes rat 3 protein as it does chick 3; many
subunit-specific anti-AChR mAbs do not cross-react between chick and
rat proteins (W. Conroy and D. Berg, unpublished results). Some, but
not all, anti-AChR mAbs previously tested on receptors reconstituted in
artificial membranes were able to influence single channel properties
(Blatt et al., 1986). If intracellular dialysis with appropriate
subunit-specific mAbs proves to be widely applicable for selectively
targeting receptor subtypes in situ, it may prove powerful
for correlating individual receptor species with unique functional responses.
The intracellular mechanism by which the mAbs block receptor function
is of considerable interest. Possibly the antibodies simply occlude the
ion channel from the cytoplasmic side. Alternatively, the antibodies
may prevent a conformational change required for channel opening.
Extensive evidence suggests muscle AChRs undergo such conformational
changes (Karlin and Akabas, 1996), and a recent report indicates that
the large cytoplasmic loop influences channel function (Milone et al.,
1998). Another possibility is that the mAbs may prevent an
intermolecular interaction such as receptor phosphorylation or linkage
to a cytoskeletal element required to optimize receptor functionality.
Future experiments will explore these possibilities.
The finding that rat intracardiac ganglion 7-AChRs behave quite
differently from rat hippocampal and PC12 7-AChRs invites speculation. It is possible that the rat 7 gene product can combine with other as yet unidentified subunits to produce heteromeric receptors with different properties. The rat 7 gene product can coassemble with muscle AChR subunits under some conditions when coexpressed in Xenopus oocytes (Helekar et al., 1994), but
it has not been shown to do so with any of the known AChR gene products in vivo. No new rat AChR gene products have been identified
since 9, and 9 is non-neuronal with a very limited pattern of
expression. Nonetheless, if an AChR gene were primarily confined to
expression in subpopulations of autonomic neurons, it could well have
escaped detection. Similar considerations apply to hypotheses based on 7 splice variants generating receptors with different properties.
A different kind of explanation is that the functional differences
among rat 7-AChR populations may be produced by cell-specific or
location-specific regulatory interactions. The diverse functions currently attributed to 7-AChRs (McGehee et al., 1995; Gray et al.,
1996; Zhang et al., 1996; Coggan et al., 1997; Fu and Liu, 1997; Ullian
et al., 1997) could certainly necessitate complex regulatory options.
Perhaps the most likely mechanism is one using some form of second
messenger-mediated receptor phosphorylation (Huganir and Greengard,
1990). Alternatively, receptor interactions with the cytoskeleton and
associated molecules could alter receptor function, as suggested above.
Intracellular dialysis of neurons via the patch pipette should provide
a means for manipulating the cytoplasmic milieu and testing several of
these hypotheses.
A final issue is the significance of 7-AChRs for signaling through
intracardiac ganglia. Approximately one-half of the intracardiac ganglion neurons sampled by single-cell RT-PCR were shown to have an
7 transcript (Poth et al., 1997), and, in approximate agreement, ~80% of the neurons tested here displayed Bgt-sensitive ACh
responses. It is not known which neuronal subpopulations in the ganglia
such cells comprise, but they are likely to include the large principal neurons that innervate cardiac muscle directly.
If intracardiac ganglion 7-AChRs retain the high relative
calcium permeability observed for 7-AChRs elsewhere (Bertrand et
al., 1993; Seguela et al., 1993), their resistance to desensitization could enable them to have a major impact on calcium-dependent events in
the neurons. Changes in intracellular calcium, for example, modulate
the firing patterns of intracardiac ganglion neurons (Allen and
Burnstock, 1987). Such changes in neuronal firing properties may be a
mechanism by which integration of neuronal signals and coding of
information occur in mammalian intracardiac ganglia (Cuevas et al.,
1997). A portion of the receptors may be destined for presynaptic sites
on the cells. In this case a slow rate of desensitization coupled with
a high relative calcium permeability could have a major effect on
modulation of transmitter release by the receptors, a role advanced
previously for them in other systems (McGehee et al., 1995; Gray et
al., 1996; Coggan et al., 1997). Either of these effects could enable
7-AChRs to be a major contributor to the neural circuit that exerts
local control over cardiac function.
| |
FOOTNOTES |
Received June 8, 1998; revised Sept. 18, 1998; accepted Oct. 5, 1998.
This work was supported by the National Institutes of Health Grants NS
12601 and 35469 and by TRDRP Grant RT65-0050. J.C. is a
University of California President's Fellow. We thank Dr. Jon
Lindstrom (University of Pennsylvania, Philadelphia, PA) for generously
supplying monoclonal antibodies.
Correspondence should be addressed to Dr. Darwin K. Berg, Department of
Biology, 0357, University of California, San Diego, 9500 Gilman Drive,
La Jolla, CA 92093-0357.
Dr. Cuevas's present address: Department of Pharmacology and
Therapeutics, University of South Florida College of Medicine, Tampa,
FL 33612-4799.
| |
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Top
Abstract
Introduction
