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Introduction |
nAChRs are ligand-gated ion
channels activated by the neurotransmitter ACh and are distributed
throughout the peripheral and central nervous system (Clarke et
al., 1985
; Wada et al., 1989; Dineley-Miller and
Patrick, 1992; Séguéla et al., 1993; Rubboli et al., 1994). To date, a gene family encoding 11 nAChR
subunits has been identified (Elgoyhen et al., 1994; for a
review see Sargent, 1993). We and others have cloned nine human nAChR
subunits: 
2, 3, 4, 5, 6, 7, 
2, 3 and 4
(Elliott et al., 1996; Fornasari et al., 1990;
Chini et al., 1992; Anand and Lindstrom, 1990; Tarroni et al., 1992; Doucette-Stamm et al., 1993; Peng
et al., 1994; Willoughby et al., 1993). The
stoichiometry of recombinant nAChRs expressed in Xenopus
oocytes is thought to be (x)2(y)3 (Anand et al., 1991; Cooper et al., 1991); however, in
recombinant expression systems 7, as well as 8 and 9 can form
functional homooligomeric receptors (Couturier et al., 1990;
Gerzanich et al., 1994; Elgoyhen et al., 1994).
Pharmacological and functional studies of recombinant rat and chicken
nAChRs expressed in Xenopus oocytes have revealed a large
diversity among the different subunit combinations (Luetje and Patrick,
1991; Connolly et al., 1992; see Sargent, 1993 and Papke,
1993 for review). Recent reports on the functional characterization of
h7 (Peng et al., 1994; Gopalakrishnan et al.,
1995) and h42 nAChRs (Gopalakrishnan et al., 1996)
indicate that the human homologs are also pharmacologically and
functionally diverse.
Many different subtypes of nAChRs have been reported in a variety of
neurons; nAChRs are present in both pre- and postsynaptic structures in
the rodent and chick central nervous system (reviewed by Sargent, 1993
and Clarke, 1995). One approach to gain insight into the molecular
composition of native nAChRs has been to compare their functional and
pharmacological profiles with those observed using recombinant
receptors. The molecular composition of some native chick and rat
nAChRs has been proposed based on their pharmacological profile and the
characteristics of their macroscopic currents (Mulle et al.,
1991; Alkondon and Albuquerque, 1993; Covernton et al.,
1994; Zhang et al., 1994). However, at the single-channel level, a good correlation has not yet been established between native
and recombinant nAChRs tested to date (Connolly et al., 1995, for reviews see McGehee and Role, 1995; Sargent, 1993 and Papke,
1993).
Administration of nicotinic agonists to rodents increases locomotor
activity and enhances learning and memory, as shown in several
behavioral tests (Clarke and Kumar, 1983; Levin et al., 1993). In human neurodegenerative disorders such as Alzheimer's and
Parkinson's diseases, there is a significant reduction in nAChR number
(Rinne et al., 1991; Nordberg, 1994), and administration of
nAChR agonists may ameliorate many of the motor and cognitive deficits
associated with these diseases (Baron, 1986; Newhouse et
al., 1988) and other motor disorders, such as Tourette's syndrome (Moss et al., 1989). More recently, a missense mutation in
the hnAChR subunit 4 was found to be associated with a form of
familial frontal lobe epilepsy (Steinlein et al., 1995).
Identification and characterization of the hnAChR subtypes involved in
these phenomena may therefore be critical in the development of
subtype-specific nAChR modulators for therapeutic purposes.
Human nAChR subunits 2, 3, 4, 5, 6, 7, 2, 3 and
4 show 91-99% amino acid identity to their rat homologs in their extracellular amino terminal domain (Anand and Lindstrom, 1990; Fornasari et al., 1990; Chini et al., 1992;
Doucette-Stamn et al., 1993; Willoughby et al.,
1993; Peng et al., 1994; Elliott et al., 1996).
These differences in the deduced amino acid sequences may affect the
properties of nAChRs: substitution of a single amino acid residue in
the extracellular amino terminal region of 3 (Hussy et
al., 1994) and 7 (Galzi et al., 1991) nAChRs subunits has been shown to dramatically affect the pharmacology of
recombinant nAChRs. Studying the properties of hnAChRs using heterologous expression may therefore provide valuable insights into
the composition, function and pharmacology of native hnAChRs. We now
report that, when expressed in Xenopus oocytes, recombinant hnAChRs display unique sensitivities to nAChR agonists and antagonists, and the pharmacology of some of these hnAChRs differs from that reported for their rat homologs.
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Methods |
Clones.
The hnAChR subunits 2, 3, 4-2, 2, 4
and 7 were cloned from cDNA libraries prepared from human brain and
the human IMR32 neuroblastoma cell line (Elliott et al.,
1996). GenBank access numbers for the cDNA nucleotide sequences are
U62431-U62439 (2-7 and 2-4, respectively). The 5
untranslated region of 2, 4-2, 2 and 7 cDNA was removed and
replaced with a Kozak consensus ribosomal binding site, 5-GCCACC-3
(Kozak, 1987). The cDNAs were subcloned into different expression
vectors, as indicated in Elliott et al. (1996), except that
the KE2RBS insert was subcloned into a pCMV vector modified by the
insertion of a T7 promoter. In vitro transcripts were
prepared using MegaScript T7 or SP6 capped RNA transcription kits
(Ambion, Inc., Austin, TX).
Xenopus oocyte injection.
Xenopus laevis frogs
were purchased from Nasco (Fort Atkinson, WI) or Xenopus One
(Beverly Hills, FL). Mature females were anesthetized by immersion in a
0.15 to 0.3% tricaine methanesulfonate solution and oocytes were
surgically removed. The follicular cell layer was partially removed
after incubation for 2 to 3 hr. in a solution containing (in mM): NaCl
(100), KCl (2), MgCl2 (1), HEPES (5) and 1.5 mg/ml
collagenase A. Defolliculation was completed manually in most cases.
Oocytes were injected with 10 to 50 nl containing 10 to 100 ng of
combinations of hnAChRs subunits X + X in vitro synthesized RNA. Pair-wise subunit
combinations were injected at a 1:1 ratio. After injection, oocytes
were incubated at 19°C for 3 to 5 days in a solution containing (in
mM). NaCl (77.5), KCl (2), CaCl2 (1.8), MgCl2
(1), HEPES (5), Na-Pyruvate (5), with penicillin/streptomycin (10 ml/liter).
Drugs.
ACh, NIC, CYT, DMPP, d-Tubo, MEC, atropine and
collagenase A were purchased from Sigma Chemical Co. (St. Louis, MO).
DHE was purchased from Research Biochemicals International (RBI,
Natick, MA). Stock solutions of agonists and antagonists were prepared and frozen. Individual aliquots were thawed and diluted in standard Ringer at the desired final concentrations.
Recording procedures.
Oocytes were examined for functional
expression 2 to 5 days after RNA injection using a two-electrode
voltage-clamp protocol, with a GeneClamp 500 (Axon Instruments, Foster
City, CA), or an Oocyte Clamp OC-725B (Warner Instrument Corp., Hamden,
CT) amplifier. Axotape and pCLAMP software (Axon Instruments), Origin
(Microcal, Northampton, MA) and Prizm (Graphpad, San Diego, CA)
software were used for data acquisition and analysis. Membrane
potential was held at either -80 mV (for partial agonist DRCs and for
antagonist experiments) or -60 mV (for full agonist DRCs); experiments
were performed at room temperature (19-23°C). Microelectrodes were filled with a 3 M KCl solution (2-4 M
resistance). The
extracellular recording solution (standard Ringer's) contained (in
mM): NaCl (115), KCl (2.5), CaCl2 (1.8), HEPES (10),
atropine (0.001), pH 7.3. Perfusion solutions were gravity fed into the
recording chamber (Warner Instruments, capacity: 110 µl) at a rate of
10 to 13 ml/min and were extruded at the opposite side of the chamber
by vacuum; perfusate exchange was performed manually by switching
between solenoid valves/reservoirs. Under these conditions, saturating concentrations of agonists could routinely activate currents with 0 to
100% rise-times of <200 msec, e.g., response in h7 in
figure 1. Agonists were applied for approximately 10 sec in most
experiments, although shorter (
5 sec) or longer (20 sec)
applications were also tested. Peak response amplitudes were measured
and used in the determination of agonist and antagonist properties.
Oocytes were washed in drug-free Ringer's for 3 to 10 min between
successive drug applications for agonist and antagonist studies, except
where otherwise indicated.
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Fig. 1.
Representative traces showing the current responses
to maximally effective concentrations of ACh in oocytes injected with mRNA encoding various human nicotinic receptors. Data shown in figures
1, 2, 3 were obtained from oocytes voltage-clamped at -60 mV. Of the
2-containing receptors, h32 receptors showed the fastest decay
kinetics to ACh application. Similarly, h34 receptors showed more
apparent desensitization than did h24 or h44 receptors
(bottom row). Currents recorded from h7 nAChRs decayed very rapidly
(upper right panel). Note the transient inward current observed in
h32- and h42-injected oocytes upon removal of agonist
(arrows). Maximally effective concentrations of ACh for the oocytes
shown here were 300 µM for h24 and h44 receptors, 1 mM
for h22, h34, h42 and h7 receptors and 3 mM for
h32 receptors
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Agonist DRCs were obtained using two different methods. 1) For partial
DRCs, responses were normalized to 1 µM ACh in all hnAChR subunit
combinations, except h7, normalized to 10 µM ACh. The normalizing
dose of ACh was applied several times to each oocyte during the course
of an experiment to check for desensitization; data were rejected if
responses to the normalizing dose fell below 80% of the original
response. 2) For full agonist DRCs, responses from each oocyte were
normalized to the maximal response for each agonist tested and used to
generate EC50 and nH estimates. For comparison
of relative agonist efficacies, the agonist responses for each oocyte
were normalized to the response elicited by an EC80 dose of
ACh (EC20 for h32). For full DRCs, hnAChR subunit combinations containing 4 subunits (h24, h34 and
h44) and h7 were tested in a Ringers solution containing 0.18 mM [Ca2+], to reduce the contribution of
Ca2+-activated Cl
currents (Miledi and
Parker, 1984) in agonist-induced responses. In this low
[Ca2+] Ringers solution, 2-containing hnAChRs showed
very small agonist-induced currents (
40 nA to 1-3 mM ACh), possibly
related to their sensitivity to external Ca2+ (Mulle
et al., 1992; Vernino et al., 1992; Mahaffy
et al., 1996), and therefore were tested in standard
Ringer's (1.8 mM [Ca2+]).
The sensitivity to the nAChR antagonists d-Tubo, DHE and MEC was
tested in standard Ringer's solution. Each oocyte was tested at all
concentrations indicated for the d-Tubo and DHE experiments, except
for d-Tubo on h22 and h34 nAChRs. For the latter, a different group of oocytes was tested with each antagonist dose, and
one curve was fitted to the (averaged) data points. The activity of MEC
was assessed at one dose (3 µM), due to the incomplete reversibility
of the block by this antagonist.
Data analysis.
Dose-response curves for agonists (full DRCs)
and antagonists (d-Tubo and DHE) were fitted by nonlinear regression
to the equations: I = Imax/[1 + (EC50/Ag)n] or I = Imax
Imax/[1 + (IC50/An)n] wherein
Imax = maximal normalized current response (in the absence of antagonist for inhibitory curves), Ag = agonist concentration, An = antagonist concentration, EC50 = agonist
concentration eliciting half maximal current, IC50 = antagonist concentration eliciting half maximal current, and
n = Hill coefficient. Antagonist curves were
constrained to Imax=1 and Imin= 0. For agonist
efficacy curves, Imin was constrained to 0, but
Imax was not constrained.
Concentration data (EC50 and IC50 estimates)
are shown as the geometric means ± S.D. Hill coefficient and
efficacy estimates are shown as the arithmetic mean ± S.D. For
the antagonists, IC50 values were converted to
Kb values using the Leff-Dougall (Leff and Dougall, 1993) variant of the Cheng-Prusoff equation:
Kb = IC50/((2 + ([Ag]/[A50])n)1/n 1), where
Ag is the agonist, A50 is the EC50 value for
the agonist and n = Hill coefficient.
Statistical tests.
The geometric values for the
EC50 or Kb data were tested for
significant differences between receptor subtypes using a one-way analysis of variance followed by a Student-Newman-Keuls or Dunn's test
for pairwise multiple comparisons. The Student-Newman-Keuls and the
Dunn's tests (SigmaStat, ver. 1.01, Jandel Corporation, San Rafael,
CA) provide a significance level of P < .05, but do not provide
the absolute P value; therefore the differences may be of greater
significance than stated in the text and tables. Differences in
arithmetic nH values for a given agonist between a 4-
and a 2-containing hnAChRs, or differences in geometric IC50 values between DHE and d-Tubo for each subunit
combination were tested for significance with an unpaired two-tailed
t test. The significance of differences in agonist potency
from partial DRCs (or the potency of MEC) among hnAChRs subtypes, was
tested with the Kruskal-Wallis one-way analysis of variance; followed by pair-wise multiple comparisons with the Dunn's test (SigmaStat, ver. 1.01, Jandel Corporation).
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Results |
Human recombinant nAChRs display differential sensitivities to
nicotinic agonists.
The nicotinic agonists ACh, NIC, CYT and DMPP
produced dose-dependent inward currents in voltage-clamped oocytes
expressing different hnAChRs subunit combinations. The kinetics of
agonist-induced currents were found to differ among the various subunit
combinations (figs. 1 and 2A). Of the
heteromeric nAChRs, currents generally decayed most rapidly in
h32 nAChRs; in contrast, currents elicited in h22,
h24 and h44 showed relatively little desensitization in the
continued presence of high concentrations of agonists (fig. 1).
Currents recorded from h34 decayed substantially faster than
those recorded in h24 or h44 nAChRs (figs. 1 and 2A). Responses from h7 nAChRs decayed much more rapidly than those from
any of the heteromeric nAChR subunit combinations (fig. 1). Agonist-dependent differences in the decay rate were also observed (for
example in h32: fig. 2, left panels), where a markedly faster
decay rate was observed with DMPP than with CYT.
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Fig. 2.
Dose-dependent responses elicited by
the application of the nicotinic agonists DMPP and CYT in oocytes
expressing the hnAChRs subunit combinations h32 or h34. A,
DMPP produced rapidly decaying responses in an oocyte injected with
h32 mRNA (left) but more slowly decaying responses in an
h34-injected oocyte (right). In each of these oocytes,
application of a high agonist concentration produced a rapidly decaying
response followed by a transient inward current when switching from
DMPP-containing to control medium (arrows). B, Current responses
elicited by the application of various concentrations of CYT showed
similar decay properties in oocytes expressing h32 or h34
nAChRs. As with DMPP, switching from cytisine-containing to control
medium produced a small inward current not seen at lower agonist
concentrations. A through D represent data obtained from four
different oocytes.
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Human nAChRs subunit combinations exhibited distinct sensitivities to
nicotinic agonists. Full dose response curves obtained for ACh, NIC,
DMPP and CYT are shown in figure 3; data are summarized in table 1. The rank order of potency (EC50
estimates) derived from the full dose-response curves was the following
(>indicates the significance level is P < .05 or higher, see
"Methods"): h22: DMPPNICCYTACh, DMPP > ACh;
h24: NICDMPP > CYT>ACh; h32: DMPPCYTNIC>ACh; h34: DMPP > CYTNIC>ACh;
h42: CYT>NIC>DMPP > ACh; h44:
CYT>NIC> DMPPACh and h7: DMPP > CYTNICACh.
These results show that ACh is the least potent of all agonists tested in most subunit combinations (h24, h32, h34 and
h42).
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Fig. 3.
Full dose-response curves for ACh, (-) NIC, DMPP
and CYT on recombinant hnAChRs. Current responses in each oocyte were
normalized to the EC80 (or EC20 for h32)
ACh response recorded in the same oocyte. Data points indicate the
mean ± S.E.M. of three to six oocytes. Where no error bars are
seen, they are smaller than the symbols.
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Steeper agonist dose-response curves (fig. 3) and thus higher Hill
coefficient values (table 1) were apparent in 4-containing hnAChRs,
compared to 2-containing receptors coexpressed with the same subunit. The differences in the Hill coefficients between h22 and
h24 nAChRs for ACh, NIC and DMPP were significant (P < .05). Hill coefficient values were significantly larger for ACh, DMPP
and CYT in h34, compared to h32 nAChRs (P < .05). Hill coefficient estimates from h44 nAChRs were also
significantly larger than those from h42 for ACh, DMPP and CYT
(P < .05). The large Hill coefficient for CYT on h22 nAChRs
is likely due to the low efficacy of the agonist on this subtype, which
gives the smallest maximal responses.
Marked subtype-specific differences were also apparent in the relative
efficacies displayed by these different nAChR agonists. CYT was least
efficacious at 2-containing hnAChRs (h22, h32 and
h42), in contrast to its efficacy shown on 4-containing hnAChRs, h24, h34 and h44. CYT displayed full agonist
activity only at ha7 (fig. 3; table 1). ACh was the most or among the most efficacious agonists at all hnAChR subunit combinations except on
h32 hnAChRs, where DMPP was markedly more efficacious than ACh.
A dose-dependent increase in the rate of decay of agonist-induced
inward currents was observed in all subunit combinations; this appeared
more pronounced in h32, h34 (fig. 2) and h7 nAChRs. This
increase in the apparent rate of desensitization was also accompanied
by a "rebound" inward current upon the removal of high doses of
some agonists in some hnAChRs (figs. 1 and 2, arrows). This rebound
current has been shown to be an indication of agonist-dependent
open-channel block in native (Maconochie and Knight, 1992) and
recombinant (Bertrand et al, 1992a) neuronal nAChRs.
Long-lasting nAChR desensitization is supported by the observation that
in some oocytes where an EC80 or EC20 dose of ACh was tested both before and up to 15 min after the completion of a
full agonist DRC, the current amplitude to the second ACh application
was reduced. This was more evident on h32-expressing nAChRs, but
was also observed in 4-containing hnAChRs. This long-lasting form of
desensitization was not observed in oocytes expressing h7 nAChRs.
These observations indicate that application of mid to high agonist
concentrations, such as those required to achieve saturation of agonist
DRCs, can result in desensitization and/or agonist-induced channel
block of neuronal nAChRs. Both can contaminate efficacy, potency and
Hill coefficient values; therefore, these estimates may not directly
reflect the interaction of the ligand with the nAChRs.
To address this issue, we have compared the rank order of potencies
estimated from full dose-response curves with those obtained in a
separate series of experiments from partial dose-response curves (fig.
4), similar to those reported for recombinant rat nAChRs
(Luetje and Patrick, 1991; Connolly et al., 1992; Covernton et al., 1994). These experiments were designed to test the
relative sensitivity of hnAChRs at agonist concentration ranges where
desensitization would be expected to be small (0.3 to 10-30 µM).
They also serve as a comparison to the only other study that has
compared the agonist profiles among all six pair-wise nAChRs subunit
combinations using the rat homologs (Luetje and Patrick, 1991). Data
were rejected if responses to the normalizing dose fell below 80% of
the initial response (see "Methods"). We found that the relative
potency displayed by these four agonists, in the ranges 30 µM, was similar using both methods for all hnAChRs except h24.
In h24 the relative potency of DMPP, NIC and ACh appeared
different: NICACh>DMPP with partial DRCs (fig. 4), whereas
NIC>DMPP > ACh was observed at 10-30 µM in the full DRCs
(fig. 3). Using either method, CYT elicited the largest responses at
doses 3 µM in this subunit combination. From the partial DRCs, it
is apparent that CYT is the least potent agonist at 2-containing
hnAChRs, whereas it is the most potent agonist at h24 and
h44 nAChRs. These results are similar to what has been reported
for their rat homologs and are consistent with the idea that subunits also contribute to the pharmacology of neuronal nAChRs (Luetje
and Patrick, 1991).
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Fig. 4.
Partial dose-response curves for h22,
h24, h32, h34, h42, h44 and h7 nAChRs.
Responses to the agonists ACh, (-) NIC, DMPP and CYT, were normalized
to the amplitude of the response elicited by 1 µM ACh in the same
oocyte, except for h7, where responses were normalized to 10 µM
ACh (response amplitude elicited by 1 or 10 µM ACh = 1). Each
symbol represents the mean ± S.E.M. of the responses observed in
3 to 12 oocytes. Where no error bars are seen, they are smaller than
the symbols.
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To analyze the effect of agonist-induced nAChR desensitization and/or
channel block on the Hill coefficient estimates obtained from the fits
to the full DRCs, we examined the slope of log-log plots from the
partial agonist DRCs. The slope of log-log plots of DRCs at low agonist
concentration ranges approximates the Hill coefficient (cf. Connolly
et al., 1992; Covernton et al., 1994; Cohen
et al., 1995). Using low concentrations of ACh (30 µM) to minimize nAChR desensitization and the contribution of the endogenous Ca2+-activated Cl current, we have
compared the slopes of these dose-response log-log plots among the
different hnAChR subtypes. The slopes of the ACh log-log plots were
markedly steeper for 4-containing hnAChRs (and h7) than for
2-containing hnAChRs (fig. 5). Log-log plots obtained
for the other agonists also displayed shallower slopes for nAChRs
containing the 2 subunit than those containing 4 subunits (data
not shown). The differences observed in log-log DRC slopes between
2- and 4-containing hnAChRs are in agreement with the results
obtained with the nH estimates derived from the full DRCs.
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Fig. 5.
Log-log plot of the ACh dose-response relation for
(A) 2-containing hnAChRs and (B) 4-containing and h7 hnAChRs.
Data points represent the mean ± S.E.M. of the responses
normalized to the current elicited by 1 µM ACh in each oocyte
(n = 3-10 oocytes). Regression lines were fitted
using least squares approximation to the data points. The slope values
of these lines are 0.62 for h22, 0.79 for h32, 0.53 for
h42, 1.61 for h24, 1.26 for h34, 1.11 for h44
and 2.08 for h7. Where no error bars are seen, they are smaller than
the symbols.
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Recombinant hnAChRs show a unique sensitivity to nAChRs
blockers.
We have tested the sensitivity of these recombinant
hnAChRs to the nAChR antagonists d-Tubo, DHE and MEC. Dose-response
curves for d-Tubo and DHE inhibition were constructed for each
hnAChR subunit combination; sensitivity to MEC was tested at a single concentration (3 µM; see "Methods"). The agonist and dose to test these antagonists on each subunit combination were selected on the
basis of 1) potency: the most or one of the most potent agonist was
used and, 2) magnitude of the response: a concentration eliciting a
large response, but relatively small desensitization upon repeated application (see fig. 4). The agonists and doses selected were the
following: h22: 10 µM NIC; h24: 30 µM ACh; h32:
10 µM DMPP; h34: 10 µM DMPP; h42: 10 µM ACh;
h44: 10 µM NIC; h7: 100 µM ACh.
DHE and d-Tubo reversibly inhibited agonist-induced currents in
oocytes expressing these different hnAChRs (fig. 6). The reversibility of nicotinic responses after MEC application (3 µM) was
variable. In some cells, full recovery was not observed after prolonged
(10-15 min) washout in drug-free Ringer's. A differential sensitivity
to the three antagonists was observed (figs. 7, 8, 9). Table
2 summarizes the Kb estimates
obtained from the Leff-Dougall variant of the Cheng-Prusoff equation
(Leff and Dougall, 1993), which corrects for both the potency of the
agonist used and its Hill coefficient from the agonist DRCs. The
Kb estimates for DHE and d-Tubo from the DRCs
(fig. 7) indicate that h42 and h44 nAChRs
are more sensitive to block by DHE than d-Tubo (P < .01, t test), whereas h7 (P < .01, t test)
and h34 are more sensitive to block by d-Tubo than DHE. In
contrast, no significant difference in the Kb
estimates for these two antagonists was found in h22, h24
and h32 nAChRs (P > .05). Human 44 was the nAChR
subtype most sensitive to block by DHE and d-Tubo.
Kb values for d-Tubo were significantly lower
for h44 than those of h42, h24 and h7
(P < .05). The rank order of potency of DHE was
h44>h42>h22h32h24>h34h7 (> indicates the significance level is P < .05).
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Fig. 6.
Inhibition of agonist-induced currents by nicotinic
receptor antagonists. Current responses recorded from oocytes
expressing h32 (A), h7 (B) or h44 (C) nAChRs. Traces
shown on each row are from the same oocyte. The time between each
application (control, agonist + antagonist and wash) was 5 to 10 min.
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Fig. 7.
The relative potency of the nicotinic receptor
antagonists d-Tubo and DHE differs among recombinant hnAChRs.
Dose-response curves (fitted by nonlinear regression to the Hill
equation, see "Methods") for d-Tubo and DHE on all seven
hnAChRs. Response amplitudes recorded upon the coapplication of either
of these antagonists and a nicotinic agonist (indicated on the right of each plot), were normalized to the current amplitude elicited by the
agonist alone. Data points represent the mean ± S.E.M. of the
responses observed in three to six oocytes. The difference in potency
between d-Tubo and DHE was statistically significant for h42,
h44 and h7 (P < .05, Mann-Whitney); h34 nAChRs cannot be tested for significance (see "Methods").
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Fig. 8.
The kinetics of agonist-induced currents in
h24 nAChRs are altered by coapplication with submaximal doses of
d-Tubo. Current responses elicited in an oocyte expressing h24
nAChRs by ACh in the absence (control), in the presence (arrow) and
after washout of 3 µM d-Tubo (wash). Holding membrane potential -80 mV.
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Fig. 9.
Amplitude of the responses (mean ± S.E.M.)
elicited by nicotinic agonists in the presence of 3 µM MEC as a
fraction of the response recorded in its absence (n = 3-10 oocytes/group). The nicotinic agonist used for each nAChRs
subunit combination is indicated in table 1. The sensitivity to MEC
observed in h44 is significantly different from that seen in
hnAChR indicated by an asterisk (P < .0001, Kruskal-Wallis
analysis of variance, followed by Dunn's test, P < .05,).
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The effect of d-Tubo appeared unusual on some hnAChRs. The inhibition
by this antagonist on h24 nAChRs was more dramatic at later times
after the activation of the inward current than at the initial peak
(fig. 8). This effect, observed in all six cells tested,
was noticeable at concentrations of d-Tubo of 0.3 µM and above. The
effect on the kinetics of agonist-induced responses produced by d-Tubo
is similar to that produced by MEC, but different from the effect of
DHE on this and other hnAChR subunit combinations tested. Our
observations suggest that d-Tubo may act noncompetitively at h24
nAChRs, in addition to its putative action at the ligand binding site.
d-Tubo also appeared to alter the kinetics of agonist-induced responses
on h44, but not h22 nAChRs (data not shown).
MEC (3 µM) inhibited agonist-induced responses by >80% in h24
and h44 and by 50% in h22, h42 and h7 nAChRs
(fig. 9). The sensitivity to MEC observed in h44
nAChRs was significantly more than that observed in h22,
h42 or h7 hnAChRs (P < .05 Dunn's test).
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Discussion |
We have shown that recombinant hnAChRs display differential
sensitivities to nicotinic agonists and antagonists, and that both and subunits contribute to the pharmacology of these ligand-gated
channels.
Agonist selectivity of recombinant hnAChRs.
Full agonist DRCs
were obtained for ACh, NIC, DMPP and CYT. Our results indicate that
when high agonist concentrations are used, such as those required to
reach saturation in DRCs, receptor activation can overlap with
agonist-induced receptor desensitization and/or channel block, which
can contaminate efficacy, slope and potency estimates. These phenomena
are not unique to nAChRs (Luetje and Patrick, 1991; Connolly et
al., 1992; Maconochie and Knight, 1992), but are also observed in
other ligand-gated channels (for review, see Jones and Westbrook,
1996). Results derived from full DRCs were therefore compared with
those obtained from partial DRCs. To our knowledge, this is the first
study in which the agonist pharmacology of recombinant nAChRs is
evaluated with both partial DRCs (in which agonist concentrations
tested are low to minimize receptor desensitization) and full DRCs. At
low agonist concentrations, no differences in relative agonist potency
were noted between full and partial DRCs in any of the hnAChRs, except
for h24. Also, the relatively larger nH estimates
observed in 4- compared to their related 2-containing hnAChRs
were observed both in partial DRCs and full DRCs.
An interesting observation is the evaluation of the differential
activity of CYT on 2 vs. 4-containing hnAChRs. Fully
saturating DRCs were obtained for CYT in all of the 2-containing
receptors, albeit with very low efficacy, yielding actual
EC50, nH and Imax values (see table
1 and fig. 2). The EC50s from these determinations showed
similar or higher potency than the other agonists examined on
2-containing receptors. However, when partial DRCs were constructed, CYT was seen to have a very low potency relative to other agonists at
equivalent concentrations (fig. 4). This latter observation is similar
to that reported for the rat homologs (Luetje and Patrick, 1991;
Covernton et al., 1994) and can be overlooked when the
sensitivity to agonists is evaluated from full DRCs.
A marked difference was observed in the kinetics of currents elicited
in h34 and h32 nAChRs, with currents elicited on h32
nAChRs decaying more rapidly than those recorded in h34 nAChRs.
This is in agreement with the kinetics reported for responses to
epibatidine on these hnAChRs (Gerzanich et al., 1995) and
for agonist-induced currents in rat 32 and 34 (Cachelin and
Jaggi, 1991; Cohen et al., 1995). The fast kinetics of
agonist-induced currents observed in h7 nAChRs are not different
from those reported by Peng et al. (1994) and Gopalakrishnan
et al. (1995) for h7 and for rat (Séguéla
et al., 1993) and chick 7 (Couturier et al.,
1990).
The agonist selectivity profile of h32, h34 and h7
nAChRs reported differs from that reported for their rat homologs. DMPP
is more potent than ACh in h32 nAChRs (figs. 3 and 4), whereas
the rank order of potency reported for rat 32 is
DMPP=ACh>NIC>CYT (Luetje and Patrick, 1991). However, two groups
have reported that DMPP > ACh for rat 32 nAChRs expressed
in Xenopus oocytes (Cachelin and Jaggi, 1991; Covernton
et al., 1994); the reason for this discrepancy is unclear.
NIC is more potent than ACh at h32 nAChRs (table 1), in agreement
with the recently reported rank order of potency of
epibatidine>NIC>ACh for h32 nAChRs expressed in
Xenopus oocytes (Gerzanich et al., 1995), but
different from the profile reported for the rat 32 (ACh>NIC:
Luetje and Patrick, 1991; Covernton et al., 1994). Although
the rank order of potencies for ACh and NIC agree between our work and
that of Gerzanich et al. (1995), the EC50
estimates obtained for ACh and NIC do not. Higher values were observed
in this study, compared to those of Gerzanich et al. (1995).
However, Gerzanich et al. used the pSP64T vector for
expression of h32 and h34. We examined the potency of ACh
and NIC with h2 cDNA subcloned into the pSP64T vector and observed
ACh and NIC EC50s of 1.75 ± 0.1 µM (n=3) and 0.79 ± 0.22 µM (n=3) for h22, 27.4 ± 8.1 µM
(n=4) and 21.1 ± 3.4 µM (n=4) for h32
and 1.3 ± 0.1 µM (n=3) and 0.3 ± 0.1 µM (n=3) for h42. The values that we observed for
h32 using the pSP64T vector are similar to those observed by
Gerzanich et al. for h32. The reason for the
differences with these vectors is not understood. By contrast, we did
not observe potency differences for ACh or NIC with h34 using
h4 cDNA (KE4.6) subcloned into the pCMV-T7 vector (table 1)
compared to the results observed by Gerzanich et al. using
the pSP64T vector.
DMPP is the most potent agonist at h34 nAChRs (figs. 3 and 4). In
contrast, the rank order of potency reported for rat 34 is
CYT>NIC=ACh
DMPP (Luetje and Patrick, 1991; Covernton et
al., 1994). NIC and ACh are also equipotent in rat 34 nAChRs
transiently expressed in mammalian HEK-293 cells (Wong et
al., 1995), whereas NIC>ACh at h34 nAChRs (Table 1), in
agreement with the rank order of potency of epibatidine>NIC>ACh
reported for h34 (Gerzanich et al., 1995). The
relative efficacies reported for ACh, NIC, CYT and DMPP for rat
34 also differ from the efficacies found in this study (Wong
et al., 1995). Taken together, these data indicate that the
pharmacology of h34 and h32 nAChRs differs from that of
their rat homologs.
The agonist sensitivity observed in h7 nAChRs is in agreement with
that reported for h7 expressed in Xenopus oocytes by Peng et al. (1994), but it differs from the sensitivity
reported for the rat (NIC>CYT>DMPP > ACh) (Séguéla
et al., 1993) and the chick homologs (NICCYT> ACh>DMPP)
(Bertrand et al., 1992b) in that DMPP is the most potent
agonist at h7 nAChRs. However, Gopalakrishnan et al.
(1995) recently reported an agonist pharmacology for h7 stably
transfected in HEK-293 cells that is closer to that reported for the
rat, wherein NIC is the most potent agonist. The reason for this
discrepancy is not clear; the full cDNA sequence of Gopalakrishnan
et al. (1995) for the h7 clones used has not been
published.
The rank order of potency observed for nAChR subunit combinations
h22, h42 and h44 is similar to that reported for
their rat homologs (fig. 4) (Luetje and Patrick, 1991; Connolly
et al., 1992). Furthermore, the relative sensitivity to
nicotinic agonists recently reported using a
86Rb+ efflux assay in h42 nAChRs stably
expressed in HEK293 cells (Gopalakrishnan et al., 1996) is
in agreement with our results.
Interestingly, even the minor divergence found in the sequence of the
amino terminal extracellular domain of subunits between human and
rat may contribute to the pharmacological differences observed between
some recombinant hnAChRs and their rat homologs, because a single amino
acid substitution in this region can profoundly affect the pharmacology
of recombinant nAChRs (Hussy et al., 1994; Galzi et
al., 1991). The identity between human and rat deduced amino acid
sequences in this domain is 93% for 3 and 94% for 7 subunits
(Elliott et al., 1996). Our observations with h32, h34 and h7 nAChRs suggest that the divergence in molecular structure between human and rat nAChR subunits 3 and 7 may
account for the altered pharmacological properties of their assembled multimeric receptors.
The Hill slope values we obtained for some agonists in 2-containing
hnAChRs are lower than those obtained for 4-containing hnAChRs.
Lower Hill coefficients have been reported for nicotinic agonists in
rat 32 compared to rat 34 nAChRs expressed in Xenopus oocytes (Cachelin and Jaggi, 1991; Covernton
et al., 1994; Cohen et al., 1995). It is possible
that nAChRs containing 2 subunits desensitize more rapidly than
4-containing receptors and that this desensitization accounts for
the lower Hill coefficient estimates; however, typically faster decay
rates were observed in h32 nAChRs than in h22 or h42
nAChR
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Abstract
Introduction
