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The Journal of Neuroscience, September 1, 2001, 21(17):6561-6568
The C Terminus of the Human Nicotinic 
4
2 Receptor
Forms a Binding Site Required for Potentiation by an Estrogenic
Steroid
Ken
Paradiso,
Jessie
Zhang, and
Joe Henry
Steinbach
Department of Anesthesiology, Washington University School of
Medicine, St. Louis, Missouri 63110
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ABSTRACT |
In addition to actions mediated by changes in gene expression,
steroids can directly modulate several transmitter-gated and voltage-gated ion channels. Despite numerous studies showing that steroids enhance or reduce ion channel activity, the site(s) that mediates steroid recognition is not known. To identify the regions in
which steroids bind and affect ion channel activity, we have taken
advantage of the observation that human 
4
2 neuronal nicotinic receptors are potentiated by an estrogen steroid, 17-estradiol, whereas a rat 42 receptor is not. Mutations indicate that a sequence (AGMI) at the end of the C terminus of the human 4 subunit forms a binding site required for 17-estradiol potentiation. In
contrast, ethynyl -estradiol (an oral contraceptive) potentiates both human and rat 42 receptors. A single tryptophan in the C
terminus of both the rat and the human 4 subunit is sufficient for
potentiation by ethynyl -estradiol, probably through a 
- interaction. Mutation of this tryptophan eliminates the ability of
ethynyl -estradiol to potentiate rat receptors. However, in human
receptors it was necessary to mutate both the AGMI sequence and the
tryptophan to eliminate potentiation by ethynyl -estradiol. The
findings that -estradiol requires the AGMI sequence but that a
single C-terminal tryptophan is sufficient for potentiation by ethynyl
-estradiol indicate that the C terminus forms a binding site for
these steroids. The binding site(s) for block appears to differ from
those involved in potentiation because the C-terminal sequence does not
affect block by steroids such as progesterone, and progesterone does
not competitively inhibit potentiation.
Key words:
nicotinic receptors; estrogen; steroids; potentiation; binding site; ligand-gated ion channel
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INTRODUCTION |
The physiological actions of
steroids are typically associated with initiation of gene transcription
after binding to cytosolic steroid receptors. However, steroids can
also have immediate effects that require a more direct mechanism of
action, including effects on neuronal excitability by "neuroactive"
steroids (Paul and Purdy, 1992
; Rupprecht and Holsboer, 1999). Rapid
modulation of neuronal excitability by neuroactive steroids is
particularly interesting because some of these steroids are produced in
the brain (so-called neurosteroids), either de novo from
cholesterol or by enzyme-mediated modification of another steroid
(Robel and Baulieu, 1995). Localized production and the ability to
alter ion channel kinetics suggest the possibility of selective actions
of neurosteroids in specific brain regions. For example, aromatase
P450, the enzyme that produces estradiol, is found both presynaptically
and postsynaptically (Balthazart and Ball, 1998), and RNA encoding
aromatase P450 is found in the hippocampus and the temporal and frontal
neocortex of the adult human brain (Stoffel-Wagner et al., 1999).
Estrogen has been shown to influence various ion channels (McEwen and
Alves, 1999; Valverde et al., 1999); therefore, local estrogen
synthesis could affect activity at both sides of the synapse.
Although it is thought that steroids can bind to ion channels, efforts
to locate a specific binding site have been unsuccessful. Here, we show
that 17-estradiol, an estrogenic steroid, directly potentiates
responses from the human 42 neuronal nicotinic receptor, and that
the C terminus of the 4 subunit is likely to form the binding site
necessary for potentiation.
The 42 receptor represents a major part of the total brain
content of nicotinic receptors (Flores et al., 1992), accounts for
80-90% of the high-affinity nicotine binding sites in the brain
(Marubio et al., 1999), and is diminished in the brains of patients
with Alzheimer's disease (Sugaya et al., 1990; Wevers et al., 1999).
Nicotinic receptors are involved in a variety of neurological disorders
(Kuryatov et al., 1997; Lindstrom, 1997; Paterson and Nordberg, 2000),
and increasing evidence suggests that presynaptic nicotinic receptors
have an important physiological role in modulating the release of many
neurotransmitters (McGehee et al., 1995; Gray et al., 1996; Coggan et
al., 1997; Wonnacott, 1997; Paterson and Nordberg, 2000). Therefore,
modulation of nicotinic receptors by estrogen or other compounds is
likely to influence synaptic signaling in several pathways.
We previously established that 17-estradiol, E2, inhibits a
receptor formed from a rat 42 neuronal nicotinic receptor (Paradiso et al., 2000), in contrast to the ability of E2 to potentiate human 42 receptors (Buisson et al., 1998). We explored this apparent contradiction to determine the difference between block
and potentiation and to identify the portions of the protein that are
responsible for potentiation.
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MATERIALS AND METHODS |
Synthesis and expression of cDNA encoding chimeric and
mutated subunits. cDNA constructs for human 4 (Kuryatov et al.,
1997) and 2 (Anand and Lindstrom, 1990) subunits were kindly
provided by Dr. Jon Lindstrom (University of Pennsylvania,
Philadelphia, PA) in pSP64 and were transferred to pcDNA3
(Invitrogen, Carlsbad, CA) for use. Rat 4 and 2 subunits
were kindly provided by Dr. J. Patrick (Baylor College of Medicine,
Houston, TX) (Sabey et al., 1999). Because of the high homology
between the rat and human subunits, identical restriction sites could
be found for the production of chimeras. Three sets of chimeric 4
subunits were made; for each set, one chimera was made with the rat
sequence from the N terminus to the chimeric join, whereas the
other chimera was made with the human sequence at the N terminus. The
chimera pairs were made at the following amino acid residues
(numbered for the mature protein): X1, 131/132
(AatII); X2, 296/297 (Alw44I); and X3, rat
268/human 268 or human 267/rat 269 (ScaI). The human 4 subunit was excised with HindIII and BamHI,
whereas rat 4 was excised with HindIII and
XbaI; next, the subunits were digested separately with the
specific restriction endonuclease, and fragments were purified by gel
electrophoresis. pcDNA3 was digested with HindIII and
XbaI or BamHI (as appropriate), and the opened
vector was purified. The two appropriate subunit fragments from the
subunits were then mixed with the opened pcDNA3 and ligated.
All point mutations were generated using the QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA). For reasons that are not
clear to us, it was more efficient to mutate the rat 4 construct in
pcDNA3. Accordingly, many of the mutations in the C-terminal tail
region were generated this way, after it had been shown that other
regions of the subunit did not affect potentiation (see Results). All
chimeras and point mutations were sequenced through the entire
translated region to confirm that the appropriate changes had been
introduced, and that no additional mutations were generated in the sequence.
Nicotinic receptors were expressed in Xenopus oocytes by
injecting the nuclei of defolliculated oocytes using the blind method described by Colman (1984). cDNA constructs encoding an 4 and a 2
subunit at a concentration of 200 ng/µl, mixed at a 1:1 ratio, were
used for all experiments. Nuclei were microinjected with 13.6 nl of
cDNA-containing solution into the nucleus of the oocyte. Oocytes were
incubated at 18°C for 2-4 d before biophysical recordings.
Two-electrode voltage-clamp recordings. A two-electrode
voltage clamp (Warner Instruments, Hamden, CT) was used to record currents from oocytes. Both voltage and current electrodes had resistances of ~1 M
and were filled with 3 M
KCl; silver chloride pellets were used for the bath and virtual ground.
The oocyte recording chamber had a volume of ~0.1 ml and was
continuously perfused with solution (either bath or test) at a rate of
~7 ml/min. Solutions were switched by hand, using Teflon rotary
valves (Rheodyne, Rohnert Park, CA). Because of the lipophilic nature
of the steroids used in our experiments, glass syringes, Teflon tubing,
and Teflon valves were used in the perfusion apparatus.
To prevent the activation of
Ca2+-activated chloride currents found in
oocytes, Ca2+ was replaced with
Ba2+ (Briggs et al., 1995). The following
external solution was therefore used (in
mM): 96 NaCl, 2 KCl, 1.8 BaCl2, 1 MgCl2, and 10 HEPES, pH 7.3. Acetylcholine (ACh) chloride (Sigma, St. Louis, MO) was prepared as a 1 M stock solution in ion exchange-purified
distilled water and stored in aliquots at 
20°C. Steroids
were purchased from Sigma or Steraloids (Newport, RI) or were
synthesized by the laboratory of Dr. Doug Covey (Washington University,
St. Louis, MO). Stock solutions were prepared as 10 mM steroid dissolved in DMSO and stored at room temperature
in the dark. Working solutions were made up on the day of the
experiment in external solution. The standard test concentration of 15 µM steroid resulted in a solution containing 0.15% DMSO,
but control experiments with DMSO alone indicated that this
concentration had no effect on ACh responses (data not shown) (Paradiso
et al., 2000).
Data were filtered at 20 Hz using an eight-pole Bessel filter
(Frequency Devices, Haverhill, MA), digitized at 50 Hz using a Digidata
1200 (Axon Instruments, Union City, CA), and analyzed with a PC clone
computer using pClamp6 and pClamp8 software (Axon Instruments). The
response was measured as the difference between the average response in
a 1-2 sec window including the peak of the response and the average
baseline current in an ~2 sec window before the onset of response.
The effect of steroids was determined by taking the ratio of the
response in the presence of steroid to the average of control responses
measured before and after the test application. One complicating factor
was that the amount of potentiation produced by our standard test
applications varied between batches of oocytes, with mean values from
1.6-fold to 5.9-fold increases in response in the presence of E2 on
different batches. A single-factor ANOVA test gave a p value
of 0.001 that the difference between batches was random. We do
not have an explanation for the variability. To compensate for the
variability, in some cases (as noted in Fig. 5) we compared the amount
of potentiation within a given batch of oocytes, in addition to tests
on pooled data.
Data analysis and statistical tests were done using Excel (Microsoft,
Seattle, WA) and Sigma Plot (SPSS, Chicago, IL).
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RESULTS |
Potentiation of human 42 receptors
Coapplication of 15 µM E2 with 1 µM
ACh potentiated (3.9-fold ± 1.2 SD; n = 84) the
response of human 42 nicotinic receptors expressed in
Xenopus oocytes (Fig.
1A) but did not
potentiate responses from a rat 42 receptor (Fig.
1B) (1.02 ± 0.14 SD; n = 36).
Potentiation was assessed from the peak response to coapplication of
ACh and steroid, compared with the peak response to ACh alone. It
should be noted that two forms of the rat 4 subunit are expressed in
the rat brain, 4-1 and 4-2 (Deneris et al., 1988). The forms differ only at the extreme C terminus, and the mRNAs for the two forms
are present at approximately equal levels in rat brain. For simplicity,
we refer to rat 4-1 subunits as rat 4 in this paper.
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Figure 1.
E2 potentiates responses from human 42
nicotinic receptors expressed in Xenopus oocytes but not
responses from rat 42 receptors. A, The responses
of an oocyte injected with human wild-type 4 plus 2 subunit cDNA
to an application of 1 µM ACh and then to 1 µM ACh coapplied with 15 µM E2 and
finally the recovery response to 1 µM ACh alone (~2 min
after the co-application). Preapplication of E2 did not increase the
amount of potentiation (data not shown). B, Similar
responses of an oocyte injected with rat wild-type 4 plus 2
subunit cDNA.
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As seen for other studies of potentiation of ligand-gated ion channels,
the amount of potentiation of the human 42 receptor is greater at
lower agonist concentrations (Fig.
2A,B).
Accordingly, the experiments reported below were performed using 1 µM ACh because this represents a submaximal
agonist concentration (Fig. 2A). The lack of
potentiation for the rat 42 receptor did not result from major
differences in activation of rat or human receptors, because 1 µM ACh is a low concentration for both types of
receptors (Fig. 2A). The concentration of 15 µM E2 used in Figure 1A
provides close to maximal potentiation (Fig. 2C).
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Figure 2.
Concentration-response data for ACh and E2.
A, The relative response to various concentrations of
ACh, normalized to the response to 1 mM, in the absence of
E2. Human 42, open circles; rat 42,
open triangles. Coapplication of 15 µM
E2 enhances the response from the human receptor
(filled circles). Lines simply
connect the points, whereas points show mean ± 1 SEM for three to six oocytes (derived from two batches).
B, Potentiation of the human wild-type receptor response
produced by coapplication of 15 µM E2 plus various
concentrations of ACh indicates that potentiation is greater at lower
concentrations of ACh. The dotted lines show values of 1 (no potentiation), half the observed maximal potentiation, and maximal
observed potentiation. Filled circles show data derived
from oocytes tested at multiple concentrations (3 oocytes at each
point), whereas open circles show pooled data from all
oocytes tested (36 oocytes at 100 µM ACh and 84 oocytes
at 1 µM ACh). C, The potentiation produced
by various concentrations of E2 coapplied with 1 µM
ACh indicate an EC50 value of ~10 µM for
E2. The apparent decrease between 20 µM and 30 µM E2 is not statistically significant, although it
could reflect mixed potentiation and block by E2 at high
concentrations. Filled circles show data derived from
oocytes tested at multiple concentrations (3 oocytes at 3 µM, 2 oocytes at 20 µM, and 6 oocytes at
other concentrations), whereas open circles show pooled
data from all oocytes tested (13 oocytes at 3 µM, 84 oocytes at 15 µM, and 4 oocytes at 20 µM).
The two symbols overlap at 3 and 15 µM E2.
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Potentiation and inhibition by steroids are separate and
independent processes
In addition to causing potentiation, steroids have been shown to
inhibit the responses of neuronal nicotinic 42 receptors (Bertrand et al., 1991; Valera et al., 1992; Sabey et al., 1999; Paradiso et al., 2000). Indeed, human 42 receptors expressed in
oocytes are inhibited by some steroids (Fig.
3, Table
1). Accordingly, we examined the question
of whether potentiation and inhibition might be related processes; for
example, inhibition could result from the action of an "inverse
agonist" that bound to the same site as potentiating steroids. Our
observations indicate that potentiation is distinct from
inhibition.
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Figure 3.
The onset and offset of potentiation are rapid.
The response of a single oocyte to a long application of 1 µM ACh shows a slow decline reflecting desensitization
(light trace). When a long application is made but the
solution is switched from 1 µM ACh to 1 µM
ACh plus 15 µM E2 during the application, the response
is rapidly potentiated, and on return to 1 µM ACh alone
rapidly returns to the same level as the response to 1 µM
ACh alone. In contrast, when the solution is switched to one containing
1 µM ACh plus 15 µM PROG, the response is
rapidly blocked but recovers extremely slowly on return to 1 µM ACh alone. The responses were taken sequentially, with
ACh alone first, followed by ACh plus E2 and then ACh plus PROG. The
holding current has been subtracted but the traces have not been
normalized. There apparently was a small increase in response amplitude
between the initial control application and the test applications.
Bars indicate onset and duration of drug
application.
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Table 1.
Progesterone block of the human 42 receptor in the
absence and presence of -estradiol indicates a lack of competition
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We used progesterone (PROG) as an example of an inhibitory steroid. One
difference between inhibition and potentiation is that inhibition
reversed relatively slowly (Fig. 3) (Paradiso et al., 2000). We also
found that the requirements for the steroid structures that produce
potentiation (discussed in next section) differ from those that
produce inhibition (Paradiso et al., 2000). Finally, pharmacological
experiments indicate that inhibition and potentiation are independent
processes. We examined this by coapplying an inhibiting steroid (PROG)
with a potentiating steroid (E2) and determining whether inhibition
was reduced by potentiation (Table 1). If the presence of the
potentiating steroid reduced inhibition, this would suggest that the
two mechanisms shared some parts of a pathway (perhaps as early as a
binding site). For the competition experiments, 3 µM or
15 µM PROG was preapplied for 20 sec to ensure that the
inhibiting steroid had ample time to interact with its binding site.
For either concentration of PROG there was no significant difference
between the amounts of block among the different E2 concentrations
(Table 1) (p > 0.06 for all comparisons). In an additional experiment, 3 µM PROG and 15 µM E2 were preapplied together for 20 sec to
allow interactions between steroids to reach equilibrium. The response
was then tested with 1 µM ACh plus 3 µM PROG and 15 µM
E2, and the test response was reduced to 0.22 ± 0.06 (four
cells). The reduction did not differ from that seen when only 3 µM PROG was preapplied (Table 1). Accordingly, the actions of steroids to potentiate and to block responses of the
human 42 receptor do not show competition and so are unlikely to
involve binding to the same or overlapping portions of the receptor.
However, block is a confounding factor in the analysis of potentiation.
In particular, the presence of block might be significant in
comparisons among steroids of differing structure because it is likely
that steroids that potentiate can also block to some extent. Hence,
stronger block might be observed as weaker potentiation, or vice versa.
Features of steroid structure important for potentiation
We first examined the ability of several steroids and some
estrogen receptor agonists to potentiate human 42 nicotinic
receptors. Some important basic features of steroid structure are shown
in Figure 4A, including
the ring nomenclature, with the A ring highlighted to indicate its
importance, and arrows marking the critical carbon 3 and 17 positions. The standard concentration of steroid adopted was 15 µM, because this concentration produced close
to maximal potentiation for E2 (Fig. 2C). In addition,
these steroids can precipitate out of solution at concentrations of
>20 µM; thus 15 µM
represents a reasonable test concentration while still being low enough
to minimize issues of steroid solubility.
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Figure 4.
Steroids tested for their ability to potentiate
the human 42 nicotinic receptor. The structures of the steroids
are identified by the abbreviations in the text, and values indicate
the ratio of the response to coapplication of 1 µM ACh
and 15 µM of the test compound to the response to 1 µM ACh alone in the same oocyte (±1 SD, with the number
of oocytes tested in parentheses). The stereochemistry
at all optically active centers is shown for E2 and
ent-E2; only partial stereochemistry for the other
steroids is shown, but they are identical to that for E2 at the
additional centers. A, The structure in the top
left corner shows the steroid ring backbone with the rings
labeled A-D. The A ring is in
bold, and arrows point to carbons 3 and
17 to indicate the importance of these positions. Although E2
potentiates responses, its mirror image (ent-E2) does
not. B, Steroids containing a saturated A
ring (317ED) or lacking a hydroxyl group
at carbon 3 (3MeE2 and
mestranol) do not potentiate. C,
Absence of a hydroxyl group at carbon 17 (E0,
E1) or addition of a hydroxyl group at carbon 16 (E3) reduces potentiation. D,
Potentiation occurs with an -hydroxyl at carbon 17 (E2), additional unsaturation in the B
ring ( 7 E2), or an ethynyl group added to
carbon 17 (ethynyl E2).
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The mirror image of E2 (ent-E2) (Fig.
4A) was ineffective at potentiating; therefore, the
steroid interacts with a site that recognizes the stereochemistry of
the steroid. Enantioselectivity indicates that E2 does not act by
some bulk effect on the membrane, because the physical properties of
enantiomers are identical, suggesting that E2 is binding to a protein.
Strong potentiation required an unsaturated A ring and a free hydroxyl
group at the 3 and 17 position (Fig. 4A). An A
ring-saturated analog of E2 (317ED) and the 3-methyl ether of
E2 (3MeE2) were ineffective at potentiating (Fig.
4B), as was the 3-methyl ether of 17-vinyl E2
(mestranol). Removal of the 17-hydroxyl group (E0) or replacement with
a keto group (E1) produced ineffective compounds, as did the addition
of a third hydroxyl group (E3) at carbon 16 (Fig. 4C).
Because our experiments do not separate affinity from efficacy, it is
possible that some of the structural requirements for potentiation
reflect occupancy, whereas others reflect the ability to produce potentiation.
Consistent with the structural features required for potentiation
discussed above, the steroids shown in Figure 4D
potentiated responses. A diastereoisomer of E2, 17-estradiol
(E2) potentiated responses almost as well as E2, indicating that
the presence of the 17-hydroxyl group is critical, whereas the
stereochemistry is less important (Fig. 4D). Two
other steroids were at least as effective at potentiation as E2:
17-vinyl E2 (ethynyl E2) and a steroid with a partially
unsaturated B ring (7 E2). These three steroids (Fig.
4D) contain the same critical structural features of
E2: an unsaturated A ring and free hydroxyl groups at the 3 and 17 position.
We believe that the steroids directly interact with the nicotinic
receptor and are not acting through estrogen receptors. Although the
human 42 nicotinic receptor and the nuclear estrogen receptors
share requirements for steroid structure (Anstead et al., 1997; Kuiper
et al., 1997; Wiese et al., 1997), there are some clear differences
between agonists for the nicotinic receptor and the estrogen receptor.
Specifically, for human estrogen receptors, E2 is much worse at
binding or activating than is E2 and E0, whereas E1 and E3 are
comparable with E2 in affinity and activity at human estrogen
receptors (Kuiper et al., 1997; Wiese et al., 1997). In addition, two
nonsteroidal agonists for the estrogen receptor inhibited responses
from the 42 receptor: diethyl stilbestrol (0.45 ± 0.12 SD;
n = 4) and kampferol (0.86 ± 0.09 SD;
n = 3). Accordingly, although there are similarities in
the structure-activity relationship for potentiation of the nicotinic
receptor and activation of the estrogen receptor, there are clear
differences that indicate structurally distinct estradiol binding sites
for the 42 nicotinic receptor and the estrogen receptors. The
differences in agonist selectivity combined with the speed of the
response allow us to conclude that the potentiation of the 42
receptor is not mediated by steroids acting through an estrogen receptor.
The C-terminal tail of the human 4 subunit is required for
potentiation by estradiol
The observation that the human 42 receptor is potentiated by
E2 and that the rat 42 receptor is not provided the
opportunity to define the portions of the receptor that are required
for potentiation. We first determined whether the species of origin for
the 4 or the 2 subunit was critical, by expressing all four
subunit combinations. For this work, we used a standard test
application of 1 µM ACh plus 15 µM E2
and measured the ratio of the response in the presence of steroid to
the response in the absence of steroid. Both receptors containing the
human 4 subunit were potentiated (human 4 plus human 2,
1.58 ± 0.36 SD; n = 11) (human 4 plus rat
2, 2.08 ± 0.34 SD; n = 6), whereas receptors
containing the rat 4 subunit were not (rat 4 plus human 2,
0.78 ± 0.09 SD; n = 3) (rat 4 plus rat 2,
0.77 ± 0.04 SD; n = 4). Accordingly, our
attention focused on the differences between human and rat 4 subunits.
We produced three pairs of chimeric subunits between the human and rat
4 subunits, with joining points located in the N-terminal extracellular region (in the cysteine-cysteine loop), at the
C-terminal end of the M3 membrane spanning region, and in the middle of
the main cytoplasmic loop. In each case, potentiation mapped to the C-terminal portion of the human 4 subunit (data not shown). We noted
that the rat and human 4 subunit differ at the extreme C-terminal
region, in the extracellular "tail" that follows the M4 membrane
spanning region. Accordingly, we constructed subunits in which only the
last two (rat, PPWLAAC) or three (human,
PPWLAGMI) residues were exchanged. This small region was
sufficient to transfer potentiation to the rat 4 subunit and remove
it from the human 4 subunit (Fig.
5A). It is interesting to note
that there is no evidence that any other nonhomologous portions of the
4 subunits have a significant role in producing potentiation,
because the potentiation observed was the same regardless of the nature
of the subunit preceding the tail (Fig. 5A). We examined the
possibility that the terminal cysteine of the rat 4 subunit acted to
prevent potentiation, for example by disulfide bond formation (DiPaola et al., 1989). However, deletion of cysteine or replacement with isoleucine or serine did not confer potentiation (Fig.
5B).
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Figure 5.
Mutations in the C-terminal tail of the 4
subunit affect potentiation by E2. Oocytes were injected with an
4 construct and wild-type 2 subunit. The first
column shows the C-terminal amino acid sequence for each
construct with the first entry (r, rat;
h, human) indicating the origin of the sequence before
the C terminus of the subunit. The sequence follows the predicted end
of the M4 membrane spanning region, with the proline pair
(PP) aligned vertically. The critical four residues
are shown in bold, and the locations of mutations are
boxed. The next column briefly describes each construct,
followed by the n value. The bar graph to
the right shows the mean ratio of the response to 1 µM ACh plus 15 µM E2 to the response to
1 µM ACh alone in the same oocyte. The vertical
lines at 1 and 3.85 indicate the average response from rat (no
potentiation) and human 42 receptors. Error bars indicate the
SEM. *indicates a p < 0.05 that the difference
between the ratio for each construct and the ratio for human wild-type
receptor, obtained from the same batch of oocytes (see Materials and
Methods), arises by chance (evaluated using the t test
assuming unequal variances); (*) is the same as *, except data from
these three constructs were compared with the pooled data from all
human wild-type receptors because wild-type responses were not
determined from the same batch of oocytes.
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To further determine that block and potentiation are separate, we also
tested the ability of PROG to block responses from the subunits with
exchanged C-terminal tails. As shown in Table 2, we found that the exchange had little
or no effect on block, confirming that the sites involved in
potentiation and block are distinct. None of the differences were
significant (p > 0.2 for all pairwise
comparisons). It therefore appeared that the human 4 C terminus did
not affect block, but instead contained a sequence of amino acids that
conferred potentiation.
Specific residues in the human 4 C terminus are required
for potentiation
Mutation of residues in the human 4 tail sequence revealed that
the nature of the final four residues (AGMI) was critical, whereas the
preceding two residues (WL) were less important for potentiation by
E2 (Fig. 5C). Deletion of the ultimate isoleucine removed
potentiation, whereas substitution with methionine reduced it.
Similarly, alterations of the methionine, glycine, or alanine reduced potentiation.
In contrast, mutation of leucine to alanine and mutation of tryptophan
to leucine or alanine did not reduce E2 potentiation (Fig.
5C). These observations indicate that these residues are unlikely to interact strongly with E2.
Not only the nature but the position of the AGMI residues is critical.
For convenience, we will speak as though the two prolines at the end of
the M4 region define a fixed position with respect to the membrane and
the rest of the receptor. Moving the AGMI sequence out from the
prolines by inserting an alanine or moving it in toward the prolines by
deleting the tryptophan removed potentiation (Fig. 5D).
These observations demonstrate that the presence of AGMI as the
terminal sequence, in and of itself, is not sufficient to confer
potentiation. However, the relative position of AGMI with respect to
the prolines is not the only critical feature, because extension of the
tail by adding one or two residues after the isoleucine also reduced or
removed potentiation (Fig. 5D). Both prolines were mutated
to glycine, with the thought that increased flexibility might result in
a structure that retains potentiation. However, this mutation also
reduced potentiation (Fig. 5D), perhaps because the rigid
bend introduced by the prolines is required for correct positioning of
the tail. These observations are consistent with the idea that the tail
occupies a relatively confined volume and must be able to adopt a
particular conformation to allow recognition of E2. It is possible
that reduced potentiation found with the relatively small changes of
alanine to glycine or glycine to alanine (Fig. 5C) also
reflects a tight binding pocket requiring a specific conformation.
The C-terminal tail sequence acts as a steroid binding site
We think that the tail is most likely to be involved in
recognition of the steroid. The data summarized in Figure 5 do not rule
out the possibility that the AGMI sequence is required for conformational changes involved in transducing the binding of E2
into the observed potentiation. However, an additional observation provides strong evidence that C-terminal residues interact directly with the steroid. We found that ethynyl E2 potentiates rat 42 receptors (Fig. 6), which is unique among
the steroids we have tested (Paradiso et al., 2000). For the rat
42 receptor, this steroid has an EC50 value
that is between 3 and 10 µM (data not shown), and 15 µM yields ~75% of the potentiation seen with 30 µM (the highest concentration tested).
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Figure 6.
Mutations in the C terminus of the 4 subunit
differentially affect the ability of steroids to potentiate responses
to ACh. The horizontal axis indicates the sequence of
the C terminus of the 4 subunit, injected with the wild-type 2
subunit. The bars indicate the response to 1 µM ACh and 15 µM steroid (E2 or ethynyl
E2) compared with 1 µM ACh alone. Error bars indicate
the SEM, with the n value indicated above each bar. The
dotted line at the value of 1.0 indicates no
potentiation.
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We reasoned that the unsaturated ethynyl moiety (C
C) might interact
with the aromatic group in the tryptophan side chain in the C-terminal
tail. Accordingly, we mutated the tryptophan to leucine to remove the
electrons while retaining the hydrophobic nature of the side chain.
The mutation was made in both the human (WLAGMI to LLAGMI) and rat tail
(WLAAC to LLAAC). We then compared the abilities of E2 and ethynyl
E2 to potentiate receptors containing the constructs. As shown in
Figure 6, receptors containing 4 subunits that had a tail with
sequences of either WLAGMI or LLAGMI were potentiated by both E2 and
ethynyl E2, as expected based on our previous results, because the
AGMI sequence is present. In contrast, a tail sequence of WLAAC
supported potentiation only by ethynyl E2 but not E2. Finally,
LLAAC did not support potentiation by either steroid, consistent with
it lacking both the tryptophan and the AGMI sequence.
The absence of steroid potentiation in the LLAAC construct could also
be explained by an increase in steroid block. In fact, application of
E2 to receptors containing the W to L mutation produced a 26%
reduction in the peak response (Fig. 6). To further address this, we
tested the ability of progesterone to block the LLAAC construct and
compared this with block of wild-type rat receptors. Progesterone was
coapplied with ACh to match the protocol for application of
potentiating steroids. At 3 µM, progesterone reduced the
peak response of rat 42 receptors by 43% (±0.08 SD;
n = 6) and that of LLAAC-containing receptors by 45%
(±0.09 SD; n = 4). At 15 µM,
however, progesterone reduced the peak response of rat 42
receptors by 63% (±0.04 SD; n = 6) and that of
LLAAC-containing receptors by 76% (±0.06 SD; n = 4).
Although there is a small increase in the ability of high
concentrations of progesterone to block the LLAAC receptors, this small
difference is not enough to account for the complete loss of
potentiation by ethynyl estradiol.
These results from Figure 6 demonstrate that the AGMI sequence is not
absolutely required for potentiation, because ethynyl E2 can
potentiate a receptor with the WLAAC tail sequence. Similarly, the
tryptophan residue (W) is not required for potentiation, because both
E2 and ethynyl E2 can potentiate receptors with the sequence LLAGMI. Our interpretation of these results is that both E2 and ethynyl E2 interact with the AGMI sequence. However, the vinyl group
of ethynyl E2 can also interact with the tryptophan. Accordingly, to
remove binding of ethynyl E2 it was necessary to remove both sites
of interaction from the protein.
On the basis of these data, it appears that the steroid rings and/or
the ethynyl group bind to the 4 C terminus, leaving the two hydroxyl
groups of the steroid free to hydrogen bond to amino acids elsewhere in
the protein. The data indicate that the C terminus acts as a binding
domain, although additional regions of the receptor are likely
necessary for the conformational changes required for potentiation.
| |
DISCUSSION |
The binding site involved in steroid potentiation differs from that
for inhibition
17-Estradiol and some structurally similar steroids potentiate
responses of human 42 receptors, whereas many other steroids inhibit responses. Our work indicates that steroids must bind to
different regions of the receptor to have these two divergent actions.
In pharmacological experiments, progesterone produces the same level of
block for control responses and for responses potentiated by 3 or 15 µM E2, indicating that PROG and E2 must bind to
nonoverlapping regions of the receptor to produce block or
potentiation. In addition, mutations of the 4 subunit have shown
that the regions of the receptor that are important for potentiation do
not affect block. Hence, although block and potentiation both involve
binding of steroids, the C-terminal amino acids that are essential for
potentiation are not necessary for block. Previous studies of
GABAA (Zaman et al., 1992) and NMDA (Park-Chung
et al., 1997) receptors have also found that inhibition and
potentiation by steroids are independent processes.
Steroid recognition by the C terminus of the 4 subunit
Our observations were made using a physiological assay, and
potentiation reflects both steroid binding and the ensuing
conformational changes that result in potentiation. Our data indicate
that the C terminus of the 4 subunit is involved in steroid
recognition and that the specific residues have less of an effect on
the subsequent conformational changes. The most compelling evidence for
this conclusion is the finding that the tryptophan in the C terminus has a specific role in recognizing the 17 ethynyl moiety
(CC) in ethynyl E2, probably through a - interaction
between the benzene ring of the tryptophan and the ethynyl group. We
think that ethynyl E2 interacts with two portions of the C terminus, both the tryptophan and the terminal tetrapeptide AGMI, because the
presence of either allows potentiation by ethynyl E2.
17-Estradiol, in contrast, interacts only with the AGMI peptide.
Because the two oxygen atoms in the E2 molecule are only ~1 nm
apart (Anstead et al., 1997), it is possible that the C-terminal tail
adopts a sufficiently extended conformation that allows it to provide
much of the binding site for E2. It is also possible that the
4 C-terminal carboxylate group forms a hydrogen bond to one of the
hydroxyls of the steroid. However, the C-terminal tail cannot provide
the entire binding site because it does not contain residues that
specifically interact with both hydroxyl groups in E2. In the
estrogen receptor, a glutamate and a histidine interact with the 3- and
17-hydroxyls, respectively (Tanenbaum et al., 1998). It is thought that
the interaction with the hydroxyl groups is critical for activation of
estrogen receptors. In the 42 nicotinic receptor, the two
hydroxyl groups appear to be essential for potentiation (Fig. 4), and
it is possible that the hydroxyl groups are important in translating
binding into potentiation.
All of the residues that we have identified as interacting with E2
are hydrophobic. In the estrogen receptor, there are 15 hydrophobic
interactions involved in stabilizing the association between E2 and
the receptor (Tanenbaum et al., 1998), and the free energy of binding
is actually derived primarily from hydrophobic interactions (Anstead et
al., 1997). The lower apparent affinity of the nicotinic receptor would
suggest that there are fewer hydrophobic interactions between the
receptor and E2. However, the two receptors are similar in the sense
that both apparently recognize steroids containing a rigid diol with a
hydrophobic and appropriately shaped spacer region.
Estradiol is synthesized in the CNS
The high nanomolar to low micromolar levels of E2 required for
potentiation represent a high concentration relative to the concentration necessary to activate the 1 and 1 (0.1-1
nM) or 2 (10-100 nM) estrogen receptors
(Petersen et al., 1998; Hanstein et al., 1999). However, the nongenomic
actions of E2 generally require nanomolar to micromolar
concentrations (Moosmann and Behl, 1999; Valverde et al., 1999), and it
has been proposed that local production by aromatase can result in high
local concentrations of E2 (Balthazart and Ball, 1998;
Stoffel-Wagner et al., 1999). It is also possible that the relevant
concentration of E2 is the concentration found in the synaptic
membrane, which would be increased by partitioning into the membrane.
Therefore, the possibility exists that the local E2 concentration
might be significantly higher than the bulk CSF levels at sites at
which the aromatase enzyme is located. Currently, the methods used to
measure steroid levels are not capable of measuring local
concentrations of steroid.
Steroids and ligand-gated ion channels
Transmitter-gated ion channels have been shown to be one target
responsible for the rapid action of neuroactive steroids. Steroids can
potentiate or inhibit the responses of several different receptors to
transmitters, and in some cases a given steroid may potentiate one type
of receptor while inhibiting another. Two well studied examples of
steroid modulation of transmitter-gated ion channels are the
potentiation of GABAA receptors (cf. Paul and
Purdy, 1992) and the inhibition of NMDA receptors (Park-Chung et al.,
1997). Although many studies have been made of the structural features
of the steroid that enhance or reduce activity on a transmitter-gated channel, there has been no success to date in identifying the regions
of any receptor that interact with steroids (Rick et al., 1998; Blanton
et al., 1999). Studies of chimeric subunits have suggested that the
ability of the anesthetic steroid alphaxalone to potentiate the
GABAA receptor requires regions of the receptor located between the N terminus and the middle of the second
transmembrane spanning region (Rick et al., 1998). In that study,
however, it was not possible to distinguish between the parts of the
receptor involved in steroid recognition and those regions required for conformational changes. The steroid promegestone inhibits the muscle-type nicotinic receptor found in Torpedo electric organ, and
photolabeling experiments have shown that it interacts with residues in
the M4 membrane spanning helix (Blanton et al., 1999). However,
promegestone is an inhibitory steroid, and our data indicate that
inhibition and potentiation of the 42 nicotinic receptor by
steroids does not involve an overlapping binding site.
We show a direct interaction between the human 42 neuronal
nicotinic receptor and -estradiol by locating a specific region of
the human 42 receptor that is necessary for potentiation of
responses by estradiol steroids. This work represents the first example
of a specific region required for steroidal effects on an ion channel,
reveals an important role for the C terminus of ligand-gated ion
channels, and suggests an additional mechanism for some of the
nongenomic effects of estrogen.
| |
FOOTNOTES |
Received May 9, 2001; revised June 21, 2001; accepted June 26, 2001.
This work was supported by National Institutes of Health Grants P01
GM47969 and R01 NS22356 (J.H.S.). J.H.S. is the Russell and Mary
Shelden Professor of Anesthesiology. We thank Dr. Douglas F. Covey for
providing steroids and for advice on their use and effects, G. Akk for
comments during the studies, and J. Bracamontes for advice on molecular biology.
Correspondence should be addressed to Dr. Kenneth Paradiso, Department
of Anesthesiology, Washington University School of Medicine, 660 South
Euclid Avenue, St. Louis, MO 63110. E-mail: paradiso{at}morpheus.wustl.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
