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The Journal of Neuroscience, October 15, 1998, 18(20):8198-8213
Two Distinct Nicotinic Receptors, One Pharmacologically Similar
to the Vertebrate  7-Containing Receptor, Mediate Cl Currents in
Aplysia Neurons
JacSue
Kehoe1 and
J.
Michael
McIntosh2
1 Laboratoire de Neurobiologie, Ecole Normale
Supérieure, Paris 75005, France, and 2 Departments of
Psychiatry and Biology, University of Utah, Salt Lake City, Utah 84112
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ABSTRACT |
Ionotropic, nicotinic receptors have previously been shown to
mediate both inhibitory (Cl-dependent) and excitatory (cationic) cholinergic responses in Aplysia neurons. We have used
fast perfusion methods of agonist and antagonist application to
reevaluate the effects on these receptors of a wide variety of
cholinergic compounds, including a number of recently isolated and/or
synthesized  toxins [-conotoxin (CTx)] from
Conus snails. These toxins have been shown in previous
studies to discriminate between the many types of nicotinic receptors
now known to be expressed in vertebrate muscle, neuroendocrine, and
neuronal cells. One of these toxins (CTx ImI from the
worm-eating snail Conus imperialis) revealed that two
kinetically and pharmacologically distinct elements underlie the
ACh-induced Cl-dependent response in Aplysia neurons:
one element is a rapidly desensitizing current that is blocked by the
toxin; the other is a slowly desensitizing current that is unaffected
by the toxin. The two kinetically defined elements were also found to
be differentially sensitive to different agonists. Finally, the
proportion of the rapidly desensitizing element to the sustained
element was found to be cell-specific. These observations led to the
conclusion that two distinct nicotinic receptors mediate Cl currents in
Aplysia neurons. The receptor mediating the rapidly desensitizing Cl-dependent response shows a strong pharmacological resemblance to the vertebrate -bungarotoxin-sensitive,
7-containing receptor, which is permeable to calcium and mediates a
rapidly desensitizing excitatory response.
Key words:
nicotinic receptor; acetylcholine; 7; chloride; Aplysia; -conotoxin ImI; suberyldicholine; methyllycaconitine; -bungarotoxin; strychnine; dihydro- -erythroidine
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INTRODUCTION |
At least two nicotinic responses,
differentiated by both their pharmacological and ionic properties, have
been described in Aplysia neurons (Tauc and Gerschenfeld,
1962 ; Kehoe, 1972a,b). One is an excitatory response involving a
nonspecific cationic channel (Ascher et al., 1978a); the other is an
inhibitory response resulting from the gating of a Cl channel (Kehoe,
1972a).
The Cl-dependent inhibitory response was found to be blocked by
-bungarotoxin (BTx) (Kehoe et al., 1976), tubocurarine (TC) (Tauc
and Gerschenfeld, 1962; Kehoe, 1972b), dihydro--erythroidine (de), and strychnine (Kehoe, 1972b) and to be elicited by
suberyldicholine (D6) (Ger and Zeimal, 1976; Kehoe, 1979). On the other
hand, the cationic response was found to be blocked, among other
compounds, by TC and hexamethonium (Tauc and Gerschenfeld, 1962), both
of which were shown to block the open channel (Ascher et al., 1978b). From these pharmacological findings, parallels were drawn (Kehoe, 1979)
(1) between the suberyldicholine-sensitive Aplysia receptor mediating the Cl-dependent response and the vertebrate nicotinic receptor found in muscle and (2) between the Aplysia
receptor mediating the cationic response and the vertebrate receptor
mediating the fast EPSP in autonomic ganglion neurons.
Since that period, thanks to the use of single-channel recording,
molecular biology techniques, and a more extensive battery of
pharmacological tools, a vast number of vertebrate nicotinic receptor
subtypes have been distinguished. The experiments described here used
fast perfusion methods and an enlarged battery of cholinergic compounds
to reevaluate the similarities between molluscan nicotinic receptors
and the many newly defined vertebrate receptor subtypes. Among the
antagonists used were -toxins from predatory Conus snails
[-conotoxin (CTx)]. These toxins have been extremely useful in
discriminating between vertebrate nicotinic receptors. In particular,
we tested the recently synthesized (McIntosh et al., 1994) CTx
ImI from the worm-eating snail, Conus imperialis. This toxin was shown to block BTx-sensitive neuronal receptors from
both mouse and rat (McIntosh et al., 1994; Pereira et al., 1996) and,
when tested on nine recombinant nicotinic receptors expressed in
Xenopus oocytes (Johnson et al., 1995), to block, selectively, the two BTx-sensitive homomeric receptors, 7 and 9. CTx ImI was also shown to block the ACh response in frog muscle (McIntosh et al., 1994) but to be only weakly active or without
effect on fish (McIntosh et al., 1994) or mammalian (Johnson et al.,
1995) muscle. The only BTx-resistant receptor that CTx ImI has
been shown to block is that mediating fast excitatory synaptic
transmission in B neurons of the frog sympathetic ganglion (Tavazoie et
al., 1997).
In the present study we have determined that the ACh-induced,
BTx-sensitive, Cl-dependent response in Aplysia neurons
can be separated into two kinetically distinct currents mediated by two
pharmacologically distinct receptors. One of those receptors shows a
strong pharmacological resemblance to the BTx-sensitive, 7-containing (Orr-Urtreger et al., 1997), calcium-permeable (Galzi et al., 1992; Seguela et al., 1993) receptor that mediates a rapidly desensitizing cationic response in vertebrate neurons.
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MATERIALS AND METHODS |
Experimental preparation. The experiments described
in this paper were performed on cells from either the buccal or pleural ganglia of Aplysia californica. The ganglia were prepared as
previously described (Kehoe, 1985). For experiments evaluating the
cationic ACh response, identifiable, unpigmented small cells from the
right pleural ganglion (Ascher et al., 1978a) were used. For
experiments on the Cl-dependent cholinergic response, individually
identifiable cells (B3, 6, 8, 9, or 10) of the buccal ganglia (Gardner
and Kandel, 1977) were used, as were cells from two distinct cell groups (medial and posterior; Kehoe, 1972b) of the pleural ganglia.
Electrodes, voltage clamp, recording procedures, and treatment of
data. All recordings were made in voltage clamp with hand-pulled microelectrodes made from a de Fonbrune microforge. For a description of the electrodes and the characteristics of the two-electrode voltage
clamp, see Kehoe (1985). Continuous recordings were made on a Servogor
340 paper recorder and on a digital audio tape recorder. Records of
currents elicited by agonist applications were digitized and sampled
on-line on a Dell computer, via a Cambridge Electronic Design 1401 interface, using the Whole Cell Electrophysiology program from
Strathclyde Electrophysiological Software.
Most of the experiments presented here concern agonist-induced
increases in Cl conductance. Illustrations of many of those experiments
only show the outward current obtained at holding potentials less
negative than ECl (e.g., between  25 and 40
mV). Although often not shown, records were made at higher holding potentials (at ECl as well as at potentials at
which the response is inverted; for example, see Fig. 1) to establish
that the drug-induced changes observed in the Cl-dependent response
were neither voltage-dependent nor attributable to a change in
ECl.
Fast perfusion application of agonists and antagonists. The
fast perfusion system used in these experiments is a modification of
that described by Johnson and Ascher (1987). In the experiments reported here, three hand-pulled, thick-walled glass tubes, held together horizontally by heat shrink tubing, delivered either artificial seawater (ASW) or agonist- or antagonist-containing solutions to the soma of the cell under study. For these experiments, the tubes were pulled to obtain an opening of each tube that was usually on the order of 75-100 µm. The so-called "control
tube," which contained only ASW or antagonist-containing ASW,
delivered a constant flow of solution over the cell under study except
for the brief period during which one of the agonist-containing tubes was stepped (by a computer-controlled stepping motor) in front of that
cell. Agonist then flowed over the cell for a brief (2 sec) period. The
rest of the time, the control tube (from which ASW constantly flowed)
was positioned in front of the cell, the agonist tubes were stepped
aside, and the flow of agonist from those tubes was blocked by a
solenoid-driven, computer-controlled valve. The usual procedure was to
apply the agonist once per minute, and when two agonists were compared,
the agonists were alternated; hence 2 min elapsed between the
application of a given agonist. However, in a few experiments in which
the control response appeared to diminish with repeated applications,
the interapplication interval was set at 90 sec or 2 min.
With only a few exceptions, antagonists were applied exclusively
through the control tube (for exceptions see relevant text and figure
legends). When concentration-response curves were established, antagonists were applied from the lowest to highest concentrations without intervening washes. A given concentration of antagonist was
maintained until the blocking effect of the antagonist appeared to
reach a plateau (usually 3-4 min). However, with certain compounds [in particular, methyllycaconitine (MLA), BTx, and strychnine] this exposure time proved to be inadequate for obtaining a maximum blocking effect, so a few experiments were performed using a single concentration with longer exposure times (see Fig.
4A'-E' and Results).
In addition to the ASW continuously flowing from the control tube onto
the cell under study, a constant superfusion of the entire ganglion
ensured the renewal of the bathing medium and the removal of the
briefly applied agonists or antagonists from the chamber.
ASW and drug-containing solutions. The ASW contained 480 mM NaCl, 10 mM KCl, 10 mM
CaCl2, 50 mM MgCl2,
and 10 mM Na-HEPES, pH 7.8. All agonists and antagonists
used were dissolved directly in ASW. BTx and the -conotoxins were
prepared at 10 µM concentrations and were further diluted
as needed. Unused 10 µM toxin solutions were frozen and
kept for later experiments. At most a small diminution was noted in the
efficacy of the toxins thus stored in solution.
Drugs. All -conotoxins were supplied by JMM.
CTx ImI, CTx MII, and A-CTx EIVA were synthesized as
previously described (McIntosh et al., 1994; Cartier et al., 1996;
Jacobsen et al., 1997), and CTx PnIA and CTx PnIB were
synthesized using methods described for CTx MII (Cartier et al.,
1996). MLA was obtained from Research Biochemicals (Natick, MA), and
all other drugs were obtained from Sigma (St. Louis, MO).
Experimental n. Each conclusion concerning the effect of an
agonist or an antagonist on a given receptor type was drawn from a
minimum of three experiments and was usually corroborated by many
more.
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RESULTS |
Although in some Aplysia cells, only one type of
cholinergic receptor is expressed, and the response to ACh therefore
reflects the increase in a single type of conductance (e.g., cationic), in other cells, more than one receptor type is present on the cell
membrane, and ACh consequently elicits a multicomponent, multiconductance response; e.g., in the medial cells of the pleural ganglion ACh elicits both Cl- and K-dependent responses (Kehoe, 1972b),
whereas both cationic and Cl-dependent responses are elicited in the B7
neurons of the buccal ganglia (Gardner and Kandel, 1977). From one
animal to the next, ACh consistently gives the same type(s) of
conductance change(s) in a given identifiable cell.
The results discussed in this paper concern cells in which ACh induces
only an increase in Cl conductance or only an increase in cationic
conductance. First, a description will be given of the kinetics of the
Cl-dependent response as observed with fast perfusion techniques,
followed by an analysis of its pharmacological properties, including
the effects on the Cl-dependent response of CTx ImI. A similar brief
evaluation will then be made of the ACh-induced cationic response. The
effects of a number of other -conotoxins on both the Cl-dependent
and cationic responses will then be described, before terminating with
a summary, derived from the data presented here, of the pharmacological
profiles of the Aplysia nicotinic receptors.
The ACh-induced increase in Cl conductance
Description of pure Cl-dependent responses to fast perfusion
application of ACh in identified cells
Among the cells belonging to the poorly defined heterogeneous
"posterior" groups of the left and right pleural ganglia are many
cells that respond to ACh with a pure Cl-dependent response (J. Kehoe,
unpublished data). ACh also activates a pure Cl-dependent response in a
number of identifiable cells of the buccal ganglia (e.g., B3, 6, 8, 9, and 10; Gardner and Kandel, 1977). However, the kinetics of the
Cl-dependent response to a fast perfusion application of ACh is
different for these different cell types. Figure
1A shows the response
to a 2 sec ACh application that is typical of certain so-called
posterior neurons: the Cl current declines rapidly to baseline before
the end of the 2 sec ACh pulse. In contrast, the responses in the
buccal cells identified above are characterized by a non-null current
that is maintained throughout the ACh application. The relative level
to which the ACh-induced current declines is, furthermore,
cell-specific (Fig. 1B,C, respectively). This
sustained current is of relatively small amplitude in B10 cells but is
very prominent in B3 cells. It is technically difficult to give a
meaningful quantitative evaluation of the proportion of rapidly
desensitizing to sustained current in the different cell types. By its
very nature the rapidly desensitizing element is probably never
maximally expressed, and the dimensions of the perfusion system and its
placement relative to the cell, which are forcibly not the same from
experiment to experiment, are important determinants of the maximal
amplitude of the desensitizing element and, hence, of its proportional
contribution to the total response. Furthermore, the proportion of the
two different elements varies as a function of ACh
concentration probably also because of the differential
desensitization of the two elements. Despite these technical
limitations, it is clear that consistent qualitative differences can be
noted concerning the kinetic characteristics of the Cl-dependent
response in different cell types. Under the conditions used here, the
sustained current in cells B3 and B6 accounts for between 50 and 75%
of the total Cl current activated by ACh at 100 or 200 µM, whereas in some neurons of the pleural ganglion no
sustained current can be detected (Fig. 1A) at any ACh concentration. In other cell types (e.g., buccal cells B10 and B9
and the medial cells of the pleural ganglion), a sustained element
exists but is less dominant than in the B3 and B6 cells.
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Figure 1.
Cl-dependent responses as a function of holding
potential in three different cell types: 2 sec application of 200 µM ACh. Comparing A (posterior neuron and
pleural ganglion), B (buccal cell 10), and
C (buccal cell 3), it can be seen that for different
cell types the Cl-dependent response shows markedly different
desensitization kinetics over the 2 sec application of ACh. In all
three cells, the response on one side of ECl
is the mirror image of the response on the other side of
ECl; the dashed lines
represent the responses measured in the three cells at 70, 75, and
70 mV, respectively, which have been inverted and superimposed on the
responses taken at holding potentials less negative than but
equidistant from ECl (solid
lines).
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No voltage dependence of the change in conductance can be detected in
the responses illustrated in Figure 1, because the responses of a given
cell taken at potentials equidistant from ECl
have identical forms and amplitudes (see figure legend). Only cells with such voltage-independent conductance changes were used for experiments on the Cl-dependent response.
No experiments were specifically designed to determine whether the peak
and plateau elements of the ACh-induced response showed differential
selectivity for different anions. However, it can be stated that
neither the rapidly desensitizing nor the sustained ACh-induced
Cl-dependent response was blocked by intracellular sulfate ions, which
have previously been shown (Kehoe and Vulfius, 1995) to block,
selectively, the GABA-induced Cl-dependent response in the same and
other cells.
Selective blockade by CTx ImI of the rapidly desensitizing
element of the Cl-dependent response
When the buccal cells used in the experiments illustrated above
(Fig. 1) are exposed to CTx ImI, the rapidly desensitizing element
of the Cl-dependent response to ACh (200 or 250 µM) is completely eliminated (Fig.
2A), and a
toxin-resistant, seemingly nondesensitizing element is revealed. [This
latter element will be referred to as the "sustained" response,
because over longer ACh applications, and particularly in isolated
cultured neurons (Kehoe, unpublished observations), it can be seen to
desensitize, although still at a much slower rate than does the
CTx-sensitive element.] It can also be seen in Figure
2A that adding 1 µM CTx ImI
exclusively to the ACh-containing solution had no effect on the
response. It is only when the toxin is also added to the control tube
solution, and hence bathes the cell before the ACh application, that
the rapidly desensitizing element of the response is eliminated. Inversely, no further block is obtained by adding toxin to the ACh
solution after the response has already been exposed to the toxin
flowing through the control tube. Three experiments were made in which
1 or 5 µM toxin was added to the ACh solution, and all
three confirmed that the presence of the toxin in the agonist solution
does not alter the selectivity of the blockade; consequently, in all later experiments the toxin was added exclusively to the solution in the control tube.
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Figure 2.
Selective elimination by CTx ImI of the rapidly
desensitizing Cl-dependent response in identified neurons of the buccal
and pleural ganglia (see Materials and Methods). A,
CTx ImI (1 µM) included in the ACh tube had no effect
on the response (compare response presented as a solid
line with that presented as an enhanced dashed
line). The same concentration of CTx ImI added to the
control tube (hence bathing the cell before the ACh application),
however, selectively blocked the desensitizing element of the response.
B, In another cell of the same type, increasing
concentrations of CTx ImI failed to significantly alter the
sustained element of the Cl-dependent response to 250 µM
ACh applied at 1 min intervals (records in toxin taken after a 4 min
exposure to each concentration). C, In a neuron
(posterior cell from the pleural ganglion; Kehoe, 1972a), which shows
only the rapidly desensitizing element of the Cl-dependent response, 1 µM CTx ImI completely blocks the response to 200 µM ACh. D, E, Selective
block by 1 µM CTx ImI of the rapidly desensitizing
element of the response of two identified neurons of the buccal
ganglion (B10 and B3, respectively) to 200 µM ACh. The
two cells show different, characteristic proportions of the rapidly
desensitizing and the sustained elements.
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The sustained element of the response remained essentially unaffected,
even when the toxin concentration was increased by 1 order of magnitude
to 10 µM (Fig. 2B). With more prolonged
applications of toxin (e.g., 10 min), a slight diminution (~10-15%)
in the sustained element could sometimes be noted.
In Figure 2C-E, 1 µM CTx ImI produces a
similar selective and total block of the rapidly desensitizing element
of the Cl-dependent response in the three cell types previously
described in Figure 1. The net response in the buccal cells thus
appears to be composed of a toxin-sensitive, rapidly desensitizing
element and a toxin-resistant, sustained element, each of greater or
lesser relative amplitude, depending on the cell studied.
The progressive decline of the rapidly desensitizing element of the
Cl-dependent response with increasing concentrations of CTx ImI can
be seen in Figure 3A in an
unidentified buccal neuron that has almost no sustained element. Given
that in cells having only the rapidly desensitizing element, the total
response was eliminated by 1 µM toxin (Fig.
3A), that concentration of CTx ImI was assumed to
completely eliminate the rapidly desensitizing response even in cells
in which it was accompanied by the sustained responsea hypothesis
supported by the form of the response in toxin-treated cells. Data from
a cell with both response elements are illustrated in Figure
3B. To better evaluate the effect of the toxin on the
rapidly desensitizing element in such cells, the presumably pure
sustained element seen in 1 µM toxin was subtracted from
the total response obtained in increasing toxin concentrations (from 0 to 500 nM). The resulting traces (Fig. 3B, solid
lines) represent the progressive toxin-induced decline of the
rapidly desensitizing element of that response.
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Figure 3.
Progressive diminution in the rapidly
desensitizing element of the Cl-dependent response of identified buccal
cells with increasing concentrations of CTx ImI. In all experiments,
200 µM ACh was applied at 1 min intervals, and increasing
concentrations of CTx ImI were introduced successively in the
control tube of the fast perfusion system. The cells were exposed to a
continual flow of each concentration for ~3-4 min. A,
Records from a buccal cell having only a very weak sustained element.
B, Records from a buccal cell with a prominent sustained
element. The response in 1 µM CTx ImI was subtracted
from the response at each of the other concentrations (dashed
lines) to obtain records of only the rapidly desensitizing,
toxin-sensitive responses (solid lines).
C, Data from eight experiments were used to calculate
the average reduction of the desesensitizing element of the control
response with increasing concentrations of CTx ImI (10, 20, 50, 100, and 500 nM and 1 µM). For cells having both
rapidly desensitizing and sustained responses, the amplitude of the
rapidly desensitizing element was obtained by subtracting the response
obtained in 1 µM toxin from that obtained at each of the
other concentrations. Because identical concentrations of toxin were
not used in each experiment, the n values have been
indicated in the inset. The SEs have been
indicated for each concentration, except for 500 nM, for
which only one point was obtained, and for 1 µM, which
yielded a total block (either by definition, see above, or by direct
reading of the data) of the desensitizing response in all cases. The
average IC50 for the eight experiments illustrated here was
estimated by interpolation to be 47 nM.
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Data from eight different experiments were used to calculate the
average percent reduction of the rapidly desensitizing response with
increasing concentrations of CTx ImI (10, 20, 50, 100, and 500 nM and 1 µM). Under the conditions used here,
the response to 200 µM ACh was reduced by half with 47 nM CTx ImI (determined by interpolation using a semilog
plot of the data included in Fig. 3C) and was reduced (as
defined above) to 0% by 1 µM toxin. No clear distinction
could be made between the concentration-response curves obtained from
cells showing only the rapidly desensitizing element and those obtained
from cells having both elements.
In contrast, even 20 µM CTx ImI (the highest
concentration tested) failed to reduce by half the sustained
Cl-dependent response to 200 µM ACh. Consequently, the
sensitivity of the two Cl-dependent responses to the toxin differs by
at least 400-fold.
Failure of other antagonists to distinguish between the rapidly
desensitizing and sustained elements of the Cl-dependent
response
In most experiments performed with BTx, MLA, de, strychnine,
and TC, the antagonist was applied to the cell exclusively through the
control tube. As can be seen in Figure
4A-E, both elements of
the Cl-dependent response were diminished by these five antagonists at
all concentrations used in these experiments, with the exception of 10 nM MLA, which produced a very small, but selective,
reduction in the rapidly desensitizing element. With none of these
antagonists was it possible to eliminate selectively one of the two
components of the Cl-dependent response.
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Figure 4.
A-E, Failure of MLA, BTx,
de, strychnine, and TC to discriminate between the two
elements of the Cl-dependent response of buccal cells to 200 µM ACh. The data illustrated were obtained from five
different cells. ACh (200 µM) was applied at 1 or 2 min
intervals, and the antagonists were applied, at increasing
concentrations, exclusively through the control tube. With the
exception of 10 nM MLA, all concentrations of all five
antagonists tested affected both elements of the Cl-dependent response.
A'-B', Comparison in the same cell of the block by 50 nM MLA and 50 nM BTx of the rapidly
desensitizing and sustained components of the two-component
Cl-dependent response in buccal neurons elicited by a 2 sec application
of 200 µM ACh. A complete recovery from the block by MLA
was obtained before applying BTx. C'-E', Comparison
in the same cell of the block by 20 µM de, 20 µM strychnine, and 20 µM TC of the rapidly
desensitizing and sustained components of the Cl-dependent response
elicited in a buccal neuron by a 2 sec application of 200 µM ACh. The three different antagonists were tested
successively on the same cell. A thorough wash, resulting in the total
recovery of the initial response amplitude, separated the three
experiments. In A'-E', the blocked responses, obtained
by subtraction, are represented by dashed lines.
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Differential effectiveness of nicotinic antagonists in blocking the
rapidly desensitizing Cl-dependent response
At first approximation it is possible to determine the relative
effectiveness of the different antagonists in blocking the rapidly
desensitizing Cl-dependent response from experiments using cells having
both the rapidly desensitizing and sustained elements (Fig.
4A-E). However, to better evaluate the
concentrations necessary to reduce the rapidly desensitizing component
by half, a series of experiments (not illustrated) was performed on
posterior pleural neurons or on buccal neurons showing at most a
very small sustained component (see an example of such an
experiment using CTx ImI in Fig. 3A). At least two or
three concentration-response curves were obtained in experiments of
this type for CTx ImI as well as for the five other classical
antagonists. In all experiments, 200 µM ACh was applied
for 2 sec at 1 min intervals, and the antagonist was included
exclusively in the control tube solution. A relatively clear separation
of the antagonists into three groups was revealed by such experiments:
with the short applications typical of the protocol used here, CTx
ImI, MLA, and BTx were approximately equally effective in blocking
the rapidly desensitizing response, with 40-50 nM being
the concentration required to block by half the response to 200 µM ACh. For de, strychnine, and TC, the required concentration for such a block was in the micromolar range, with TC
being the least effective.
To further define the relative efficacy of the different antagonists,
an antagonist concentration approximately equal to that necessary for
blocking the rapidly desensitizing component by half was tested
independently, i.e., not as part of a series of concentrations used for
establishing concentration-response curves, and was applied for up to
at least 8-10 min to make certain that the relatively short exposure
to the antagonist given in the concentration-response experiments did
not yield an underestimation of its blocking capacity. Thus, the effect
of 50 nM MLA on the two-component Cl-dependent response was
compared with that of the same concentration of BTx (Fig.
4A',B'), whereas the effects of de, strychnine,
and TC, at 20 µM each, were similarly compared (Fig.
4C'-E'). In these figures, the control response and the
response in the presence of the antagonist are both represented by
solid lines, whereas the difference between the control response and
that taken after exposure to the antagonist (the so-called "blocked
response") is represented by dashed lines.
Findings obtained with the experiments of the type shown in Figure
4A'-E' combined with those from experiments designed
to establish concentration-response curves confirmed that for blocking the rapidly desensitizing element of the response, CTx ImI, MLA, and
BTx are similarly effective and that both de and strychnine are
more effective than TC, with the IC50 values obtained for the latter three antagonists being ~2, 5, and 20 µM,
respectively. It can furthermore be seen by comparing Figure 4,
A and A' and B and B', that
a longer exposure to a given concentration of either MLA (50 nM) or BTx (50 nM) leads to a significantly
more complete block of the rapidly desensitizing element. Additional
experiments (data not shown) suggested that, under "equilibrium"
conditions (defined as a complete stabilization of the response
amplitude), 10 nM BTx was sufficient for blocking the
rapidly desensitizing element by half.
Differential effectiveness of nicotinic antagonists in blocking the
sustained Cl-dependent response
An indication concerning the effectiveness of most antagonists to
block the sustained Cl-dependent response is already present in the
data illustrated in Figure 4, A-E and A'-E'. As
was seen above, CTx ImI fails to block the sustained element. For
the other antagonists, however, the rank order of effectiveness is quite similar to that seen for the blockade of the rapidly
desensitizing element; i.e., MLA and BTx are more effective than
de, strychnine, or TC, and both de and strychnine are more
effective than TC (Fig. 4, compare C'-E').
In experiments such as those illustrated in Figure 4, the block of the
sustained element was estimated from the response amplitude at the end
of the 2 sec ACh application, so any "washing off" of the
antagonist that might occur during the application of the agonists
would yield an inaccurate estimate of its blocking capacity. Consequently, a series of experiments comparing the efficacy of various
antagonists was performed in which the antagonist was present first
only in the control tube solution and then in both the control and
ACh-containing solution. Only one agonist concentration was used for a
given comparison. Adding the antagonist to the ACh-containing solution
failed to enhance the block by BTx and only enhanced the block
obtained with the other antagonists by at most 20%, so the general
conclusions were not significantly affected by that aspect of the
methodology.
Differential effectiveness of nicotinic agonists in eliciting the
two Cl-dependent responses
To evaluate the relative capacity of different agonists to
activate the rapidly desensitizing and sustained Cl-dependent
responses, experiments were done in which the response to a fixed
concentration of ACh (100 µM) was compared, in the same
experiment on the same cell, to those elicited by 10, 50, 100, and
often 200 µM concentrations of another cholinomimetic.
The concentration of ACh used in these experiments was far from
maximal, because the amplitudes of both elements of the Cl-dependent
response to ACh continue to increase with increasing concentrations
well beyond the 100 µM concentration used here (data not
shown).
It was found that whereas nicotine and cytisine both preferentially
activate the rapidly desensitizing element of the Cl-dependent response, suberyldicholine preferentially activates the sustained element. In Figure 5, the responses to
increasing concentrations of cytisine and of suberyldicholine are
compared in buccal cells, which show a prominent sustained Cl-dependent
component (Fig. 5A,B), with the responses to the same
agonists in a posterior neuron (pleural ganglion), which shows only the
rapidly desensitizing response (Fig. 5C, left, right). It
can be seen that the response to cytisine is essentially identical in
the two cell types; at the concentrations used here, it consists almost
exclusively of a rapidly desensitizing element, even in cells having a
marked sustained response to ACh (Fig. 5A). At
concentrations greater than 100 µM (data not shown),
cytisine becomes less specific and, even during the 2 sec cytisine
pulse, elicits a weak sustained Cl current in cells in which ACh
elicits such a current. It should be noted that the response to 10 µM cytisine is approximately equal in amplitude to that
of the rapidly desensitizing element of the response to 100 µM ACh (estimated by subtraction of the sustained element
from the total ACh response in Fig. 5A to be 4.3 nM). The peak currents elicited by the three cytisine
concentrations are indicated in Figure 5.
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Figure 5.
Preferential activation, by cytisine and D6, of
the rapidly desensitizing and sustained Cl-dependent responses,
respectively. A, Comparison, in a buccal cell showing a
marked sustained element in the ACh Cl-dependent response, to 100 µM ACh, 10 µM D6, and increasing
concentrations of cytisine (10, 50, and 100 µM). Note
that at the concentrations applied here, there is practically no
evidence of a sustained Cl-dependent response to cytisine, and that the
response to 10 µM cytisine (4.54 nA) is equal in
amplitude to the rapidly desensitizing response to 100 µM
ACh (4.53 nA, obtained by subtraction of the sustained element from the
total ACh response). For easier evaluation of the cytisine-induced
currents, they have been reproduced in the inset with an
expanded x-axis and a reduced y-axis.
B, Responses of a similar buccal cell to 100 µM ACh and to increasing concentrations of
suberyldicholine (1, 10, and 100 µM). Note that the
sustained response to 10 µM suberyldicholine is larger
than that to 100 µM ACh. C, Responses to
increasing concentrations of both cytisine (C'; cytisine
and ACh records presented in inset with expanded
x-axis and reduced y-axis) and
suberyldicholine (C") of a buccal cell showing a
predominantly rapidly desensitizing response to 100 µM
ACh (C' and C"). Note that the responses
to all three agonists desensitize during the 2 sec application, and
that the response to 10 µM cytisine (C')
is larger than that to 100 µM ACh, whereas no response
can be detected to 10 µM suberyldicholine
(C"). The desensitizing response to 100 µM
suberyldicholine (C") is only approximately one-third
that of the response to the same concentration of ACh.
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Even at concentrations at which a 2 sec agonist pulse fails to elicit a
sustained response to the above-mentioned cholinomimetics (nicotine and
cytisine), it was possible to observe a slowly developing, low-amplitude activation of that response when either nicotine (>20
µM) or cytisine (>10 µM) was applied
continually through the control tube. This more slowly developing
background activation of the receptor mediating the sustained response
resulted in a weak diminution in the corresponding sustained element of
the ACh response elicited during the period that one of the two above cholinomimetics flowed through the control tube.
Nicotine and cytisine both induce a marked desensitizing block of the
receptor they preferentially activate (see below), with nicotine
inducing the most persistent blockade. For example, a 10 min wash was
required for the rapidly desensitizing element of the Cl-dependent ACh
response to recover its initial amplitude after a 2 sec application of
200 µM nicotine. Such a "desensitizing blockade"
explains, for example, the failure of the amplitude of the response to
cytisine to increase significantly with increasing cytisine
concentrations (Fig. 5A,C', insets).
A selective activation of the sustained element of the Cl-dependent
response, in contrast, is obtained with D6. A comparison of the records
in Figure 5B and those illustrated in Figure 5C" reveals the distinct preferential activation by suberyldicholine of the
sustained element. In Figure 5B, it can be seen that even with 1 µM suberyldicholine, a clear sustained element is
elicited, and at 10 µM the sustained response to that
agonist is considerably larger than that elicited by 100 µM ACh (compare the response at the end of the 2 sec
agonist application to 100 µM ACh with that to 10 µM suberyldicholine). In Figure 5C", it can,
in contrast, be seen that in a cell showing at most a very weak
sustained element in the ACh response, suberyldicholine elicits no
response at 10 µM, only a very weak rapidly desensitizing
element at 50 µM, and, at 100 µM, a rapidly
desensitizing response that is only approximately one-third that
elicited by the same concentration of ACh. These data were obtained
during the same experiment and from the same cell as those illustrated
in Figure 5C' comparing the responses to cytisine and
ACh.
A study of the effects of CTx ImI on the responses of buccal cells
to ACh and suberyldicholine confirms the selective action of
suberyldicholine as described above. In Figure
6A, the responses of a
buccal neuron to 100 µM ACh and to 20 µM
suberyldicholine were evaluated before and after inclusion of 500 nM CTx ImI in the control solution. In the experiment
illustrated in Figure 6A, ACh elicited both the
rapidly desensitizing and sustained elements of the Cl-dependent
response. The response to 20 µM suberyldicholine, on the
other hand, is exclusively of the sustained type and is considerably
larger than the sustained response elicited by 100 µM
ACh. CTx ImI had no effect on the suberyldicholine response, which
is consistent with the failure of low concentrations of suberyldicholine to elicit a response in cells having only the rapidly
desensitizing Cl current (Fig. 5C"). The rapidly
desensitizing element of the ACh response, on the other hand, was
completely eliminated.
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Figure 6.
Further evidence for the differentiation, by
suberyldicholine and cytisine, of two distinct Cl-dependent responses.
A, Effect of 500 nM CTx ImI on the
responses to 100 µM ACh and 20 µM
suberyldicholine of a buccal cell showing a prominent sustained element
in the two-component Cl-dependent response. Note the selective
elimination in the ACh response of the rapidly desensitizing element
and a failure of the toxin to affect the response to suberyldicholine.
The periodic rapid deflections seen in the ACh records in
A represent spontaneous synaptic activity.
B, Effect of 1 µM cytisine (applied in the
control tube only) on the ACh response of a buccal cell to 100 µM ACh and 10 µM suberyldicholine. Cytisine
can be seen to block only the rapidly desensitizing element of the ACh
response, whereas it has no effect on the response to
suberyldicholine.
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As mentioned above, both nicotine and cytisine cause a desensitizing
block of the receptor they preferentially activate (that mediating the
desensitizing Cl-dependent response). A selective but sometimes partial
block of the rapidly desensitizing element of the ACh response can
thereby be obtained by applying low concentrations of nicotine or
cytisine through the control tube. The block by 1 µM
cytisine of the rapidly desensitizing response to 100 µM ACh is shown in Figure 6B. It can be seen in the same
figure that the response to suberyldicholine, which, at 10 µM selectively activates the sustained Cl-dependent
response (Fig. 5), is unaffected by the cytisine application, as is the
sustained element of the ACh response.
The ACh-induced nonspecific cationic response
The cholinergic receptor gating the cationic response is the only
cholinergic receptor found on a small group of cells in the right
pleural ganglion (Ascher et al., 1978a). Cells from this group were
chosen for evaluating the pharmacological characteristics of that
receptor.
Effects of CTx ImI and other antagonists on the nicotinic
receptor mediating the cationic cholinergic response
The blocking effect of many antagonists (e.g., TC and
hexamethonium) acting on the receptor mediating the cationic response is highly sensitive to membrane voltage, presumably because such antagonists act only on the open channel (Ascher et al., 1978b; Slater
and Carpenter, 1982). Such a voltage-dependent blockade of the ACh
cationic response is shown in Figure
7A. As described in Materials
and Methods, in most experiments the antagonist was not included in the
agonist-containing solution. However, because of the type of blockade
obtained with many compounds on the cationic response (i.e., an open
channel block), the antagonist was introduced in both the control and
ACh solutions in these experiments. When using a sufficiently fast
perfusion, 20 µM hexamethonium (C6) flowing continually
through the control tube has no effect on the cationic response (data
not shown). It is only when hexamethonium is present after the channel
has been opened by the ligand (i.e., added to the ACh-containing
solution) that a blockade occurs. This can be seen in Figure
7A by comparing the initial amplitudes of the responses
obtained at 90 mV before and after application of ACh and C6. During
the 2 sec application of the C6-containing ACh solution, there is a
rapidly developing block of the response at 90 mV. At 30 mV, this
blockade is minimal. The blockade of the same receptor by TC was shown
to have a similar voltage sensitivity (Ascher et al., 1978b).
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Figure 7.
Effect of C6 and CTx ImI on the ACh-induced
cationic response. ACh was applied on the cell soma for 2 sec once
every minute (A, B) or once every 90 sec
(C). Between each ACh application, the holding
potential was alternately set at 30 or 90 mV. A, C6
(20 µM) present in both the control and agonist tubes
blocks the cationic response to 200 µM ACh only when the
ligand-gated channel is open, and it does so in a voltage-dependent
manner. The records taken in the presence of C6 are superimposed on the
control records in the right column (control vs C6).
Whereas at 30 mV the responses are essentially identical in the
absence (control, solid line) and presence
(C6, dashed line) of the antagonist, at
90 mV, a reduction in amplitude rapidly develops during the 2 sec
application. Note, however, that even at 90 mV the response amplitude
at the beginning of the 2 sec application is unaffected by the presence
of C6. These records were taken after 5 min exposure to C6 through the
control tube. B. CTx ImI (1 µM) blocks
the cationic response to 250 µM ACh in a
non-voltage-dependent manner. The toxin, flowing continuously and only
from the control tube, reached its maximum blocking effect within 3 min. The records shown in the middle column were taken
after ~7 min exposure to CTx ImI. In the right
column, the control records have been superimposed on the
records taken in the presence of toxin, which have been multiplied by
7.2. At both potentials, the amplified toxin records superimpose
directly on the control responses, showing that there was no
voltage-dependent element in the toxin block of the cationic ACh
response. C, Progressive and reversible block of the
cationic response to 250 µM ACh with increasing
concentrations of CTx ImI, with each increasing toxin concentration
being placed in the control tube after the stabilization of the
response amplitude at the previous toxin concentration. Ten minutes
after returning toxin-free ASW to the control tube, the response
returned to its control amplitude.
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Unlike the case with hexamethonium, the channel does not need to be
open for CTx ImI (1 µM) to block the cationic response (Fig. 7B). With CTx ImI it was unnecessary to add the
antagonist to the ACh-containing solution. Including the toxin only in
ASW flowing through the control tube was sufficient to induce a rapid, marked, and reversible block of the response that is apparent immediately on application of ACh. Again, in contrast to the block by
hexamethonium, that with CTx ImI shows no voltage dependence. This
is seen in the last set of records of Figure 7B, in which the two records taken in the presence of the toxin (at 30 and 90
mV) are both seen to superimpose on the control records when they are both multiplied by the same factor (7.2).
To establish the concentration dependence of the toxin-induced block,
increasing concentrations of toxin were applied successively through
the control tube of the fast perfusion system. One of four such
experiments is shown in Figure 7C, in which 250 µM ACh was used. The response rapidly stabilized at each
toxin concentration, and in all experiments the effect was completely
reversible after a 10-15 min wash. The IC50 value for the
experiment illustrated in Figure 7C, which was obtained by
interpolation using a semilog plot, was 160 nM. In the
three other experiments, performed using 200 µM ACh, only
three toxin concentrations were used (50, 100, and 200 nM).
The IC50 values obtained in those experiments were estimated by the same method to be ~120, 150, and 170 nM,
respectively, giving an average IC50 of 150 nM.
The concentration required for an equivalent, non-voltage-dependent
blockade by either de or strychnine (Slater and Carpenter, 1982) was
~2 orders of magnitude higher.
Failure of most cholinomimetics to activate the
cationic response
None of the selective agonists discussed above (cytisine,
nicotine, and suberyldicholine) elicited a response in the cells showing only the cationic response to ACh. This was true whether a low
concentration (1 or 10 µM) or a high concentration (200 µM) of agonist was used.
It was hypothesized that perhaps the responses were not being detected
because of an extremely rapid desensitization of the underlying
receptor by the agonist. However, no rapidly desensitizing cationic
response was revealed when the speed of the concentration jump was
increased by reducing the diameter of the fast perfusion tubes. Second,
it would be expected that if such a desensitizing response existed but
was somehow undetectable with the fast perfusion system used, it should
be possible to detect a strong "blocking" effect by the seemingly
inactive agonist, similar to that seen on the rapidly desensitizing
Cl-dependent response when 1 µM nicotine or cytisine was
applied by the control tube (see Fig. 6B). This was
not found to be the case; a 100 µM concentration of
either nicotine or cytisine was required to reduce the cationic
response by half. Suberyldicholine failed to have a significant
blocking effect on the cationic response, even at 100 µM
concentrations.
The only agonist tested (other than ACh and carbachol) that was capable
of eliciting the cationic response was dimethyl-4-phenyl-piperazinium (DMPP), and the response to 100 µM DMPP was only
~10-20% of that to an equivalent concentration of ACh. Furthermore,
DMPP was not a selective agonist, because it activated, albeit weakly,
both of the Cl-dependent responses as well.
Effects of other toxins from Conus snails (CTx MII, A-CTx
EIVA, CTx PnIA, and CTx PnIB) on the nicotinic ACh responses
CTx MII is a toxin isolated from the venom of a fish eating
Conus snail, Conus magus. It has been found to be
a highly effective antagonist on recombinant 32 receptors
(Cartier et al., 1996). 3 and 2 subunits are found in mammalian
neurons, including those of autonomic ganglia, and the antagonist
profile of the receptor mediating the fast EPSP in autonomic ganglion
neurons is similar to that of the Aplysia receptor mediating
the cationic response; therefore, CTx MII was tested on the
cholinergic Aplysia receptors to see whether it would act
selectively on the receptor mediating the cationic response. As can be
seen in Figure 8A, at 1 µM (3 orders of magnitude higher than the
IC50 value for the block of recombinant 32 vertebrate
receptors), it had no effect on the cationic response and no effect on
either element (rapidly desensitizing or sustained) of the
two-component Cl-dependent response.
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Figure 8.
Effect of CTx MII, A-CTx EIVA, CTx PnIA,
and CTx PnIB on the cationic and Cl-dependent responses to 100 or
200 µM ACh. A, B, Neither
CTx MII (1 µM) nor A-CTx EIVA (1 µM)
has an effect on either the cationic response of the unpigmented
pleural ganglion cells (A, B, left records) or on either
component of the Cl-dependent responses in buccal neurons (A, B,
right records). C, CTx PnIA (1 µM) weakly blocks the cationic response as well as the
sustained Cl-dependent response (left and right
columns, respectively) and strongly blocks the rapidly
desensitizing Cl-dependent response in the same cell (right
column). D, CTx PnIB (1 µM)
causes a small reduction in all three nicotinic responses (cationic,
rapidly desensitizing, and sustained Cl-dependent responses). No
agonist effect of any of the four toxins could be detected.
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We also tested on the Aplysia receptors a toxin belonging to
the newly defined A-conotoxin family. This toxin, A-CTx EIVA, which was isolated from the fish-eating snail Conus
ermineus, was shown by Jacobsen et al. (1997) to block, at
nanomolar concentrations, both Torpedo and mouse
1-containing muscle receptors expressed in Xenopus
oocytes but to be without effect on neuronal BTx-sensitive receptors. Because the Aplysia neuronal receptor mediating
the sustained Cl-dependent response shows some pharmacological
similarities to the mammalian skeletal muscle receptor (e.g., blocked
by BTx and activated by suberyldicholine), this toxin looked like a
possible candidate for a selective antagonist of that receptor. As can be seen in Figure 8B, A-CTx EIVA also fails to
affect any of the Aplysia ACh receptors studied.
The effects of two other -conotoxins isolated from the venom of the
molluscivorous snail Conus pennaceus were previously studied
on Aplysia neurons bearing the nicotinic receptor mediating the cationic response. Fainzilber et al. (1994) found that bath application of 0.5-1 µM concentrations of either CTx
PnIA or CTx PnIB elicited, like ACh, a depolarization. Furthermore,
they observed a desensitizing block of the depolarizing response to ACh
as long as the toxin bathed the cell. Using synthesized CTx PnIA and
PnIB, we were unable to duplicate the agonist effect observed by
Fainzilber et al. (1994) on the cells responding to ACh with a cationic
response or to observe any agonist effect on the cells responding to
ACh with a two-component Cl-dependent response. Both toxins did,
however, act as weak antagonists of these responses, as can be seen in
Figure 8, C and D. Both toxins (at 1 µM) caused a small, reversible reduction in the cationic response (Fig. 8C,D, left records). In Figure 8C,
right records, it can be seen that 1 µM CTx PnIA
almost completely blocked the rapidly desensitizing Cl-dependent
response but also partially blocked the sustained element; CTx PnIB
(Fig. 8D, right records) slightly blocked both of the
Cl-dependent elements (Fig. 8D, right column),
showing a somewhat more marked diminution of the sustained response.
Pharmacological profiles of the receptors mediating the ionotropic
responses in Aplysia neurons
From the findings reported above, three distinct "nicotinic"
receptors can be distinguished, and Figures
9 and 10
summarize the pharmacological characteristics of the three responses
they mediate: cationic, rapidly desensitizing Cl, and sustained Cl.
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Figure 9.
Relative ability of eight antagonists to block the
three types of nicotinic response to 200 µM ACh expressed
in approximate IC50 values. Estimates of IC50
values were made by visual inspection of the data and have been
established with the notion of obtaining a weighted rank ordering
rather than of obtaining rigorously defined IC50 values
(see Results). A perusal of a given tone of column
(black, gray, or white)
permits an evaluation of the differential facility with which the
different antagonists block a given response type. A comparison of the
three column tones for each antagonist reveals the ability of a given
antagonist to discriminate between the different response types. Empty
columns with two asterisks are cases in which no effect
(NE) on the ACh response was seen with the maximum
concentration tested, the value of which is indicated in lieu of the
data column. One asterisk indicates that the antagonist
can induce a slight reduction in the tagged response, but that the
highest concentration used failed to block the response by half (only
case being that of 20 µM CTx ImI on the sustained Cl
response). V-d, Instances in which the block of the
cationic response was voltage-dependent.
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Figure 10.
Relative effectiveness of different
cholinomimetics in eliciting the three different types of nicotinic
response. The log scale on which the data have been plotted shows, for
example, that under the criteria used here (see Results),
nicotine and cytisine are at least 10 times more effective than ACh in
eliciting the rapidly desensitizing Cl-dependent response, whereas DMPP
is 20 times less effective. Because of the desensitizing block
(D-b) that nicotine and cytisine induce in the
desensitizing Cl response, only low concentrations (1-20
µM) of those agonists were used in determining their
relative efficacy on that receptor. The double asterisk
indicates cases in which no response at all could be elicited
(NE, no effect) with the highest concentration tested
(200 µM).
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Antagonists
Figure 9 summarizes the results obtained using the cholinergic
anatagonists on the three nicotinic response types. The efficacy of the
antagonists, presented on a log scale, has been indicated as the
approximate antagonist concentration necessary to reduce the response
to 200 µM ACh by half (IC50). The
values included in the figure are those estimated in experiments in
which the standard protocol was used. Lower IC50 values
were obtained when longer exposures were used for evaluating the
blockade by BTx and MLA of the desensitizing Cl response (see
above). The IC50 values presented in Figure 9 are very
approximate, "rounded off" estimates obtained by visual inspection
of many experiments performed on a variety of cell types. This approach
was used in preference to a simple "greater or less than" analysis,
because it permits an easier comparison across and within response
types and offers a calibrated, weighted rank ordering. However, these
values cannot be taken as rigorously quantitative estimates of
IC50 values, for which it would have been necessary to work
always on exactly the same cell type and to use a less-variable fast
perfusion system. In some cases, the efficacy of an antagonist on a
given response was so low that no IC50 could be obtained
with the antagonist concentrations used (Fig. 9, see legend for
details).
The pharmacological profiles revealed in Figure 9 permit a very clear
separation between the cationic response and the Cl-dependent responses
and show that the separation between the two Cl-dependent responses, in
contrast, depends almost exclusively on CTx ImI.
Agonists
The agonists offer a more detailed differentiation between the
three responses, as can be seen in Figure 10. To obtain approximate estimates of relative efficacy of different agonists, the responses to
varying concentrations of a given cholinomimetic were compared with the
response obtained with 100 µM ACh. With agonists capable of inducing a desensitizing block of the activated receptor(s) (e.g.,
nicotine and cytisine on the receptor mediating the desensitizing Cl-dependent response), only low concentrations were used for such an
estimate of efficacy.
Figure 10 clearly displays the preferential activation by nicotine and
cytisine on the receptor mediating the desensitizing Cl response and
the preferential activation by suberyldicholine of the receptor
mediating the sustained Cl response. Even more striking is the relative
ineffectiveness of all agonists tested here in activating the receptor
mediating the cationic response.
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DISCUSSION |
Two pharmacologically distinct receptors mediate
Cl-dependent responses in Aplysia neurons
The dissection by CTx ImI of the ACh-induced Cl
current in Aplysia neurons into a toxin-sensitive, rapidly
desensitizing element and a toxin-resistant, sustained element and the
differential activation of these two elements by different agonists
strongly suggest that two different cholinergic receptors mediate the
two kinetically defined increases in Cl conductance. This conclusion is
reinforced by the finding that the relative proportion of the two
elements is cell-specific.
While studying cholinergic synapses in the buccal ganglion, Gardner and
Kandel (1977) noted that, in some cells, the synaptically activated or
ACh-induced Cl-dependent response diminished with repetition, whereas
in others it did not. The authors' conclusion that "rate of
desensitization is ... an additional criterion for characterizing
otherwise similar receptors for neurotransmitters" appears to receive
support from the pharmacological findings reported here.
That more than one receptor subtype for a given transmitter can mediate
a Cl conductance increase was recently demonstrated in the vertebrate
GABAergic system, in which pharmacologically and molecularly distinct
GABAA and GABAC receptors were shown to mediate
rapidly desensitizing and slowly desensitizing Cl currents, respectively (for review, see Polenzani et al., 1991; Feigenspan and Bormann, 1994; Bormann and Feigenspan, 1995; Johnston, 1996 |
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Abstract
Introduction
