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Vol. 286, Issue 1, 311-320, July 1998
5 Subunit Alters Desensitization, Pharmacology,
Ca++ Permeability and Ca++ Modulation of Human
Neuronal 3 Nicotinic Receptors1
Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Functional effects of human 
5 nicotinic ACh receptor (AChR) subunits
coassembled with 3 and 
2 or with 3 and 4 subunits, were
investigated in Xenopus oocytes. The presence of 5
subunits altered some properties of both 3 AChRs and differentially
altered other properties of 32 AChRs vs. 34
AChRs. 5 subunits increased desensitization and Ca++
permeability of all 3 AChRs. The Ca++ permeabilities of
both 325 and 345 AChRs were comparable to that of
7 AChRs. As we have shown previously, 5 subunits increased the
ACh sensitivity of 32 AChRs 50-fold but had little effect on
34 AChRs. 5 caused only subtle changes in the activation potencies of 3 AChRs for nicotine, cytisine and
1,1-dimethyl-4-plenylpiperazinium (DMPP). However, 5 increased the
efficacies of nicotine and DMPP on 32 AChRs but decreased them on
34 AChRs. Immunoisolation of cloned human AChRs expressed in
oocytes showed that 5 efficiently coassembled with 3 plus 2
and/or 4 subunits. As expected, human AChRs immunoisolated from
SH-SY5Y neuroblastoma cells showed that AChRs containing 3 and
probably 5 subunits were present, but 4 AChRs were not. In brain,
by contrast, 42 AChRs were shown to predominate over 3 AChRs.
Some of the brain 42 AChRs were found to contain 5 subunits.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Neuronal
nicotinic AChRs are thought to be formed by pentameric assemblies of
certain combinations of 2, 3, 4, 5, 6, 7, 8, 9,
2, 3 and 4 subunits (Deneris et al., 1991
; Role, 1992; Sargent, 1993; Le Novere and Changeux, 1995; Lindstrom et al., 1995; McGehee and Role, 1995; Lindstrom, 1996). The
homologous subunits of an AChR are thought to be organized around a
central cation channel like barrel staves so that parts of the M1 and M2 transmembrane domains of all subunits contribute to the lining of
the channel. In the case of muscle-type AChRs, which are known to have
their subunits organized around the channel in the order 1
1
1, there are two ACh binding sites at interfaces
between 1 and or between 1 and subunits, but the 1
subunit is not thought to contribute contact amino acids to these
binding sites (Karlin and Akabas, 1995). The stoichiometry of 42
AChRs expressed in oocytes is known to be (4)2
(2)3 (Anand et al., 1991; Cooper et
al., 1991), and it is thought that these subunits are similarly organized around the channel in the order 42422, which
results in two ACh binding sites at interfaces between 4 and 2
subunits. 3 subunits can form functional AChRs in combination with
2 or 4 subunits, and it is presumed that these also probably have two ACh binding sites. 5 is known to be a subunit of AChRs
containing 3, 4 and/or 2 subunits in chick ganglia (Conroy
et al., 1992; Vernallis et al., 1993), in a human
neuroblastoma (Wang et al., 1996), and associated with a
small fraction of the 42 AChRs in chick brain (Conroy and Berg,
1995). The stoichiometry of 5 containing AChRs has not been directly
determined. However, the observation that 5 does not form functional
AChRs when expressed in Xenopus oocytes alone or in paired
combination with 3, 2 or 4 (Wang et al., 1996)
suggests that 5 subunits, like 1 subunits, cannot interface with
the sides of these subunits that are involved in forming ACh binding
sites (Karlin and Akabas, 1995). Thus it has been suggested that 5
may occupy a position homologous to that of 1 in muscle-type AChRs
(Wang et al., 1996). For example, the order of subunits
around the channel might be 32325.
Our initial studies of human 5 subunits expressed in
Xenopus oocytes showed that they assembled efficiently with
human 3 and 2 or human 3 and 4 subunits to form AChRs that
desensitized more rapidly and that, especially in the case of
325 AChRs, exhibited altered pharmacological properties (Wang
et al., 1996). Here we extend these electrophysiological
studies in Xenopus oocytes and conduct immunoprecipitation
studies to investigate the fraction of various AChR subunits in
extracts of rat and human brain that have 5 associated with them.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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cDNAs.
The cDNA sequences for human 3 (unpublished EMBL
accession no. X53559) and 2 (Anand and Lindstrom, 1990) were
subcloned in expression vectors pcDNAI (Invitrogen, San Diego, CA) and
pSP64poly(A) (Promega, Madison, WI), respectively. The cDNA for human
5 was first described by Chini et al. (1992) and was
kindly provided by Dr. Francesco Clementi (University of Milan). It was
subcloned in the pSP64poly(A) vector. The cDNA for human 4 was
cloned in this lab from a cDNA library from the neuroblastoma cell line SH-SY5Y (Gerzanich et al., 1997). It was then subcloned into
the pcDNAI vector. 1 and cDNAs were described previously (Luther et al., 1989). Epitope tagged 5t cDNA was
described previously (Wang et al., 1996). Human , 
and
cDNAs were kindly provided by Dr. Andrew Engel (Mayo Clinic).
Expression of human 3 AChRs in Xenopus
oocytes.
cRNAs for human AChR subunits 3, 2, 4 and 5
were synthesized in vitro using T7 (if the cDNA was in the
pcDNAI vector) or SP6 (if the cDNA was in the pSP64poly(A)
vector) RNA polymerase (mMESSAGEmMACHINE, Ambion, Austin, TX).
Oocytes were prepared for microinjection as described previously
(Gerzanich et al., 1995) and injected with equal amounts
(5-15 ng) of cRNA for each of the subunits. They were incubated for 3 to 4 days after injection in media containing 50% L15 (GIBCO BRL), 10 mM HEPES buffer, pH 7.5, 10 U/ml penicillin and 10 mg/ml streptomycin
at 18°C.
Electrophysiological procedures and drug application.
Currents in oocytes were measured using a standard two-microelectrode
voltage-clamp amplifier (Oocyte Clamp OC-725, Warner Instrument Corp.,
Hamden, CT). Electrodes were filled with 3 M KCl and had resistances of
0.5 to 1.0 M
for the voltage electrode and 0.4 to 0.6 M for the
current electrode. All records were digitized (MacLab/2e interface
and Scope software (AD Instruments, Castle Hill, Australia), stored on
a Macintosh IIcx computer and analyzed using AXOGRAPH software (Axon
Instruments, Foster City, CA). The recording chamber was continually
perfused at a flow rate of 10 ml/min with saline solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.6. Atropine (0.5-1 µM) was included in all solutions
to block responses of endogenous muscarinic AChRs. Application of
agonists was performed as described in detail previously (Gerzanich
et al., 1995). In summary, all agonists were applied by
means of a set of 2-mm glass tubes directed on the animal pole of the
oocyte. Application was achieved by manual unclamping and clamping of a
flexible tube connected to the syringe with the test solution.
Typically delay between beginning of the application and first
deflection of the induced current was about 0.25 sec. The Hill equation
was fitted to the concentration-response dependencies using a nonlinear
least-squares error curve fit method (KaleidaGraph, Abelbeck Software):
I(x) = Imax[xn/(xn + EC50n)], where I(x)
is current measured at the agonist concentration x,
Imax is the maximal current response at the saturating
agonist concentration, EC50 is the agonist concentration
required for the half-maximal response and n is the Hill
coefficient.
channels, Cl-free solutions were used
for oocyte preincubation (6-12 hr) and for the perfusion during
recordings (Francis and Papke, 1996). The "normal"-Ca++
solution included 90 mM NaMeSO3, 2.5 mM KOH, 10 mM HEPES
and 1.8 mM Ca(OH)2. Additionally, 48 mM dextrose was
supplemented in the normal solution in order to yield osmolarity equal
to the "high"-Ca++ solution, which contained 18 mM
Ca(OH)2 and the same concentration of the other ions as the
"normal"-Ca++ solution. Both solutions were buffered
with methanesulfonic acid to pH 7.3. Reversal potentials of the
currents were determined either by 6-sec agonist applications at
different holding potentials or by 2-sec ramps of the holding potential
from 
50 to +50 mV during agonist application after the current
reached a steady state. Both protocols gave similar estimates for the
reversal potential. Control ramp currents obtained before agonist
applications were subtracted from the ramp currents during AChR
activation.
Purification and radioimmunoassay of AChRs from oocytes, SH-SY5Y
cells and human brain.
Purification, immunodepletion and solid
phase radioimmunoassay of AChRs from oocytes were performed as
described previously (Wang et al., 1996). AChRs from the
human neuroblastoma cell line SH-SY5Y, neocortex from post-mortem human
brain and whole rat brain tissue were isolated in accordance with the
method of Whiting and Lindstrom (1986) and Wang et al.
(1996). For radioimmunoassay, 250-µl aliquots of tissue extract
either were mixed directly with 50 µl of the mAb-Actigel and
[3H]-epibatidine (5.3 nM) or were preabsorbed with 50 µl of mAb-Actigel before mixing with [3H]-epibatidine
and a fresh aliquot of the mAb-Actigel. mAb-Actigel contained 5 mg/ml
of mAb. After 8 to 12 hr of incubation at 4°C, the Actigel was rinsed
three times with ice-cold PBS, 0.05% Tween buffer. The amount of bound
AChRs was determined by labeling with 5 nM
[3H]-epibatidine, followed by liquid scintillation
counting (Wang et al., 1996). Nonspecific binding of the
AChRs to mAb-Actigel was determined by incubation of aliquots of tissue
extracts with an irrelevant mAb or normal rat IgG-Actigel under the
same conditions.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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5 subunit enhances desensitization in recombinant human neuronal
3 AChRs.
The time course of the currents induced by saturating
concentrations of ACh in oocytes expressing AChRs after coinjection of
325 or 345 cRNA combinations are compared with those after 32 and 34 cRNA coinjections in figure
1. ACh-evoked currents reached a maximum
and then decayed biphasically, showing both a transient and a plateau
phase. Small "rebound" currents, commonly explained as channel
block by agonist, were observed only for AChRs containing 4 subunits
(fig. 1, bottom two traces). The onset of the current in the AChRs
containing 2 subunits (fig. 1, top two traces) was significantly
steeper (0.23 ± 0.1 and 0.17 ± 0.06 sec to peak for
32 and 325 combinations, respectively) compared to 4
subunit-containing AChRs (0.77 ± 0.34 and 0.43 ± 0.23 sec
to peak for 34 and 345, respectively) (bottom two
traces). Listed data represent the mean of 7 to 9 oocytes for each
subunit combination ± S.D. Resolution of the current onset for
2-containing AChRs was limited by the perfusion time (see
"Materials and Methods").
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5 subunits to the 32 combination resulted in AChRs
with notably faster desensitization. T1/2 of the
current decay upon exposure to a saturating concentration of ACh
decreased from 1.1 to 0.64 sec (fig. 1, left plot on the top panel). In addition, the amount of desensitization (percent of current from the
peak to plateau) increased from 46% to 68% (fig. 1, right plot on the
top panel). A similar phenomenon was observed when 5 subunits were
coexpressed together with 3 and 4 subunits. Both the rate of
desensitization (T1/2 of decay decreased from 1.8 to
0.7 sec) and amount of desensitization (increased from 21% to 41%)
were enhanced in 345 compared with 34 AChRs (fig. 1,
bottom panel).
5 subunit alters pharmacology of recombinant human neuronal 3
AChRs.
Pharmacological profiles of 3 AChRs were investigated
using four nicotinic agonists: ACh, nicotine, cytisine and DMPP.
Concentration-response curves for these agonists were built from data
collected from oocytes expressing four different 3 neuronal AChR
subtypes (fig. 2). Concentration-response
curves for ACh and nicotine, which are shown for comparison with the
effects of DMPP and cytisine, are from our previous study (Wang
et al., 1996). All currents were normalized to the maximal
currents induced by ACh for each AChR subtype. ACh was used for
normalization of efficacy of the nicotinic agonists because it is the
endogenous agonist. Values for the EC50, Hill coefficients
and the relative maximal responses are listed in table
1. Comparison of the families of the
concentration/response curves built for 32, 325,
34 and 345 AChRs revealed striking differences in
pharmacological properties among these AChRs.
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2 subunits for 4 subunits in 3 AChRs resulted
in decreases of potency for ACh, nicotine and DMPP (table 1).
Furthermore, this resulted in increased efficacy of nicotine, changing
it from a partial to a full agonist. Efficacy for cytisine also
increased from 23% to 56% with no significant changes in apparent
affinity. In addition, concentration-response curves for the agonists
tested had higher Hill coefficients for 34 AChRs than for
32 AChRs.
Notable changes in pharmacological properties were observed when 5
subunits were added to 32 AChRs (fig.
3; table 1). Thus, as we have shown
previously (Wang et al., 1996), 325 AChRs had
almost 50 times higher sensitivity to ACh compared with 32 AChRs.
Less significant increases of apparent affinity were observed for
nicotine and DMPP. In contrast, efficacies of these agonists changed
dramatically, nicotine switching from a partial (55%) to a full
agonist (Wang et al., 1996), and DMPP increasing in efficacy
from 107% to 187% compared with ACh. This, in essence, converted ACh
and nicotine into partial agonists.
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5 subunits to 34 AChRs caused less significant
changes in apparent affinities for the agonists tested (fig. 2; table
1). Only cytisine exhibited a moderate increase of apparent affinity
for 345 AChRs compared with 34 AChRs, and there was
basically no change in the rank order of potencies of agonists. In
contrast to 32 AChRs, where addition of 5 subunits increased the efficacy of DMPP to greater than that of ACh, addition of 5 to
34 AChRs decreased the efficacy of DMPP from 100% to 13%. Overall, concentration-response curves for 345 AChRs had
higher Hill coefficients than curves built for 325 AChRs
(table 1).
5 subunits enhance Ca++ permeability and
Ca++ modulation of recombinant human neuronal 3
AChRs.
Relative permeability of Ca++ through AChRs was
evaluated by the shifts of reversal potential caused by changes in
extracellular Ca++ concentration. More precise estimates of
the permeability ratios were constrained by our inability to monitor
intracellular cation concentrations while using the two-electrode
voltage-clamp method. 7 AChRs were shown previously to have
exceptionally high permeability for Ca++ ions, comparable
to that of NMDA receptors (Bertrand et al., 1993; Seguela
et al., 1993; Castro and Albuquerque 1995; Delbono et
al., 1997). In contrast, muscle AChRs have rather low
Ca++ permeability (Vernino et al., 1992; Dani
and Mayer 1995; Francis and Papke 1996). These two AChRs were used to
"calibrate" the range of the extracellular
Ca++-dependent shift of reversal potential (fig.
4) and, subsequently, to compare the
relative Ca++ permeabilities of 3 AChR subtypes. Human
7 AChRs exhibited a 17.8 ± 0.9 mV (n = 12)
positive shift of reversal potential as a result of a 10-fold increase
of Ca++ concentration from 1.8 to 18 mV. Human muscle AChRs
formed from 1, 1, and 
subunits exhibited a shift of only
0.8 ± 0.9 mV (n = 4). 32 and 34 AChRs
had similar shifts of reversal potential upon increase of
Ca++ concentration (5.8 ± 0.8 mV (n = 7) and 6.1 ± 1.2 mV (n = 6), respectively). This
suggests similar contributions by both 2 and 4 subunits to the
AChR channel lining. Incorporation of 5 subunits in both
325 and 345 AChRs dramatically increased the
Ca++-dependent shift of the reversal potential to 13.7 ± 1.4 mV (n = 11) and 11.7 ± 1.1 mV
(n = 10), respectively. This indicates that the
Ca++ permeabilities of human 325 and 345
AChRs approach that of homomeric 7 AChRs.
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3 AChRs (fig. 4).
Although for 34 AChRs this increase of amplitude could be
attributed solely to the increase in the driving force due to the
change of the reversal potential upon increase of the Ca++
concentration, for 32 AChRs, the increase of amplitude in 18 mM
Ca++ was 3-fold larger. Addition of 5 subunits increased
the 32 AChR-mediated current, whereas no increase was observed
for 34 AChRs. Thus 2 and 4 subunits clearly contributed
differently to extracellular Ca++ modulation of 3 AChRs,
and 5 further enhanced this modulation for 325 AChRs.
Evidence that 5 subunits can assemble in AChRs with four
different subunits.
Neurons frequently express 3, 2, 4
and 5 subunits (e.g., Conroy and Berg, 1995; Wang
et al., 1996). Coinjection of equal amounts of all four
subunit cRNAs
3, 2, 4 and 5resulted in AChRs that
responded to ACh application in a distinct manner. The time course of
activation and desensitization of currents from 3245 AChRs
(fig. 5) resembled most closely the time
course of 345 AChRs (fig. 1), though current rise and decay
were both slower. Higher concentrations of ACh were required in order
to saturate the response, and a small rebound current was observed upon
removal of 3 mM ACh (fig. 5, left). The
concentration-response curve yielded a satisfactory fit with a two-site
Hill equation. The higher-affinity site (S1), with an
EC50 of 24 µM, constituted ~35% of the maximal
response. The lower-affinity affinity site (S2), with an
EC50 of 345 µM, constituted ~65% of the maximal response. DMPP behaved as a partial agonist with a maximal response equal to 65% of the response induced by the maximal concentration of
ACh. Efficacy of DMPP for the 3245 subunit combination did
not match efficacies for other double and triple subunit combinations tested (table 1). S1 for DMPP (~45% of all sites) had an
EC50 of 3.3 µM. S2 (~55% of all sites) had
an EC50 of 110 µM. The S2 site detected by
both ACh and DMPP differed in EC50 from those observed for
these agonists on 32, 34, 325 and 345
AChRs.
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5 into 3 AChRs
expressed in oocytes, we immunoisolated [3H]-epibatidine
labeled AChRs with subunit-specific mAbs (fig. 5). Precise evaluation
of the composition of the AChRs formed in these conditions was
constrained by the availability of mAbs. mAb210 crossreacts with both
human 3 and human 5 AChR subunits (Wang et al., 1996).
The efficiency of 5 subunit incorporation into 3 AChRs was
estimated by an mAb 142 epitope-tagged 5 subunit (Wang et
al., 1996) termed 5t. A specific mAb is not
available for human 4 subunits.
Virtually all [3H]epibatidine binding sites were absorbed
by the 2-specific mAb290 (Peng et al., 1994) from oocytes
expressing 3, 2 and 5 subunits (fig. 5), and virtually none
from oocytes expressing 3, 4 and 5 subunits (fig. 5). When all
four subunits were expressed, more than 85% of the AChRs were found to
contain 2 subunits. Efficiency of 5 coassembly with 3 and 2
subunits was 65%, and with 3 and 4 subunits was about 50% (fig.
5). When all four subunits were expressed, more than 70% of the AChRs
contained 5 subunits (fig. 5). Hence, when all four (3, 2,
4 and 5) AChR subunits are expressed in oocytes, the majority of
AChRs contain 3, 5 and 2 subunits. The differences in
expression levels of 325 (~10 fM/oocyte) and 345
(~2 fM/oocyte) AChRs (fig. 5) did not allow for evaluation of
efficiency of the incorporation of 4 subunits when all four subunits
were expressed in oocytes.
Analysis of subunit composition of native human AChRs using
mAbs.
We used the available mAbs to assay incorporation of 5
subunits in AChRs from neuronal tissues of central and peripheral origin. The human neuroblastoma cell line SH-SY5Y expressing
postsynaptic type 3 AChRs (Wang et al., 1996) was used as
a model of ganglionic type AChRs. Post-mortem human brain tissue from
neocortex was used to characterize central 3 AChRs. For comparison,
the expression level of 4 AChRs was evaluated using mAb299 (Peng
et al., 1994). Quantities of the different AChRs
immunoisolated in the same experiment are compared for both tissues in
figure 6.
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3 AChRs predominate in SH-SY5Y cells, and about half of these
contain 2 subunits. mAbs were not available with which to determine
independently the fraction of these AChRs that contain 5 or 4
subunits. As expected, no 4 AChRs were found.
Most (63%) of the human neocortex extract AChRs that contained 2
subunits also contained 4 subunits. Of these 42 AChRs, 36%
may also contain 5 subunits because they could be adsorbed by
mAb210.
In order to evaluate the relative amounts of various AChR
subtypes in whole brain, we performed a similar immunoisolation of
[3H]epibatidine binding sites from extracts of complete
rat brains. As in human neocortex, the major
[3H]-epibatidine binding component was adsorbed by both
mAb299 to 4 and mAb290 to 2 (fig. 6), which confirms that
42 is the dominant central neuronal AChR with high affinity for
epibatidine. About 20% of these 42 AChRs appeared to have 5
associated with them, because they could be preadsorbed with mAb210.
The amount of 3 or 5 AChRs in this tissue was about 4% of the
42 AChRs. Most or all of these appeared to contain 2 subunits,
but this measurement was difficult because so few 3 AChRs were
present.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Our results prove that, when expressed in Xenopus
oocytes, human 5 subunits are efficiently incorporated with 3 and
2 or with 3 and 4 subunits to form AChRs that differ in both
dose dependence of activation and cation channel properties from AChRs containing only 3 and 2 subunits or 3 and 4 subunits. These results suggest that 5 subunits alter channel properties because they contribute directly to structure and can alter the
EC50 or efficacy of some agonists. Although they may not be
part of the structure of the agonist binding sites, the 5 subunit
contribution to the overall structure of the AChR influences the
ability of the AChR to make the concerted changes in subunit
orientation or conformation that are required for channel opening or
desensitization.
It was shown recently that chick 5 subunits can efficiently assemble
together with 4 and 2 subunits to form AChRs with distinct
properties (Ramirez-Latorre et al., 1996).
Immunoprecipitation studies have shown that only a minor fraction of
native chick brain 42 AChRs contain 5 subunits (Conroy and
Berg 1995). In contrast, a majority of native 3-containing AChRs, at
least in autonomic ganglia, are thought to have 5 subunits
incorporated (Conroy et al., 1992; Vernallis et
al., 1993; Conroy and Berg 1995). Thus determination of the
functional impact of 5 subunit on 3 AChRs is crucial to
understanding the physiological contributions of individual subunits to
native "ganglionic-type" neuronal nicotinic AChRs.
Pharmacology.
When 5 is coexpressed with 3 and 2
subunits, two types of AChRs may be formed: 32 and 325.
As we have shown previously by immune precipitation and have confirmed
here, in these conditions more than 70% of the 3 AChRs contain 5
subunits (Wang et al., 1996). The presence of 5 subunits
produces a uniform change in functional properties.
Concentration-response curves for the 325 subunit combination
do not resolve two subpopulations of AChRs. EC50 for ACh
differs 50-fold between 32 and 325 AChRs. Additionally, the efficacy of DMPP changed dramatically between these two subunit combinations. DMPP had significantly higher efficacy (183%) than ACh
for 325 AChRs. Higher efficacy of DMPP compared with ACh was
reported previously for rat 32 and 34 AChRs expressed in
the Xenopus oocytes (Cachelin and Jaggi, 1991). Oddly,
however, when rat 34 AChRs were transiently expressed in HEK-293
cells, DMPP was reported to behave as a partial agonist with less than 30% efficacy compared with ACh (Wong et al., 1995).
Overall, DMPP exhibited remarkable sensitivity to the human AChR
subunit combination expressed. Despite only moderate changes in
EC50 for the four 3 AChRs tested, DMPP exhibited large
differences in efficacy. DMPP had only 13% efficacy for 345
AChRs, was as efficacious as ACh on 34 AChRs, was slightly more
efficacious than ACh on 32 AChRs and was almost twice as
efficacious as ACh on 34 AChRs. This characteristic of DMPP could
prove useful in identification of the subunit composition of native
human 3 AChRs.
3 AChRs tested.
It had higher efficacy (50% for 4-containing AChRs than for
2-containing AChRs (20%). This difference in efficacy for cytisine
between 2- and 4-containing AChRs was also observed for rat 3
AChRs (Papke and Heinemann, 1993). However, for rat 34 AChRs
transiently expressed in the HEK-293 cells, cytisine behaved as a full
agonist compared with ACh (Wong et al., 1995).
Of the four subunit combinations tested, concentration-response curves
built for AChRs containing 4 subunits compared with AChRs containing
2 subunits were significantly steeper, with Hill coefficients closer
to 2 for all agonists but cytisine. This could reflect the slower
desensitization rates observed for 4-containing AChRs, which could
permit better resolution of responses at high agonist concentrations.
Alternatively, the presence of a subpopulation of AChRs with different
agonist affinity could modify the slopes of concentration-response
curves. Covernton et al. (1994) reported significantly
higher Hill slopes in Xenopus oocytes for rat 34 AChRs
than for 32 AChRs.
Desensitization.
For both 325 and 345 AChRs,
rates and magnitude of desensitization were higher than for 32
and 34 AChRs. Addition of the rat 5 subunit to 42 has
also been reported to cause acceleration of desensitization
(Ramirez-Latorre et al., 1996). Enhancement of
desensitization in 5-containing AChRs might be expected to shift
EC50 values for activation to higher concentrations. However, increases of apparent affinity for ACh and nicotine were observed when 5 subunits were added to 32 AChRs. Thus the
pharmacological effects of 5 subunits probably do not reflect
changes only in rates of desensitization.
32 and 34 AChRs indicates that switching of
2 for 4 structural subunits significantly influences both the
kinetics and the pharmacological properties of the AChRs. Similar
phenomena were described previously for heterologously expressed chick
and rat 3-containing AChRs (Luetje and Patrick, 1991; Papke, 1993;
Hussy et al., 1994; Gerzanich et al., 1995; Fenster et al., 1997). It was suggested that 2 and 4
subunits contribute directly to the ligand binding pocket on the
interface with subunits. This raises a question of the possible
position of the 5 subunit in the 3 AChR pentamer and the
mechanisms by which 5 might influence functional properties.
Pentameric structure of 3 AChRs is assumed on the basis of homology
within the gene family and from comparison of the sizes of AChRs
obtained in sucrose-gradient experiments (Wang et al.,
1996). The inability of 5 subunits to assemble directly with 3 or
subunits to form functional AChRs, together with lack of 5
influence on the ligand affinities in the equilibrium binding
experiments (Wang et al., 1996) suggests that 5 subunits
do not contribute to the ligand binding pocket at the interface between
3 and subunits. This indicates that changes in the macroscopic
kinetic properties and pharmacological profiles of 325 and
345 AChRs observed electrophysiologically are determined not
by the 5 subunit's direct interaction with agonists but by the
overall conformational changes that it induces in AChRs. In addition,
an 5 subunit present in an AChR would be expected to contribute
one-fifth of the amino acids lining the cation channel and thereby
potentially affect ion flow directly.
Ca++ permeability and modulation.
Native and
recombinant 7 AChRs were shown to have Ca++
permeabilities comparable to that of NMDA receptors (Bertrand et
al., 1993; Seguela et al., 1993; Castro and
Albuquerque, 1995). Previously it was shown that native and recombinant
rat 3 AChRs have significant Ca++ permeability (Fieber
and Adams, 1991; Adams and Nutter, 1992; Vernino et al.,
1992; Rogers and Dani, 1995). Dependence of the reversal potential on
extracellular Ca++ indicates that human 32 and
34 AChRs could conduct a significant amount of Ca++
ions. Because of the much slower desensitization rates of 3 AChRs
compared with 7 AChRs, 3 AChRs could potentially, over prolonged
periods, conduct more Ca++ than could 7 AChRs. Moreover,
introduction of 5 subunits further increases the Ca++
permeability of 3 AChRs, producing, after a 10-fold increase of
extracellular Ca++, a shift of the reversal potential
comparable to that of 7 AChRs. This suggests that 352 and
354 AChRs may play more important roles than previously
suspected in ACh-induced Ca++-mediated effects in both the
peripheral nervous system and the CNS.
3 AChRs are directly involved in synaptic transmission from
preganglionic neurons. Ca++ ions entering neurons through
postsynaptic AChRs during EPSCs were shown to trigger a
Ca++-dependent K+ current (Tokimasa and North,
1984). In the CNS, presynaptic nicotinic AChRs were shown to exert
facilitatory effects by increasing presynaptic Ca++
concentration (Mulle et al., 1992).
Potentiation by extracellular Ca++ of recombinant and
native AChRs is viewed as an important mechanism of modulation (Mulle et al., 1992; Vernino et al., 1992; Amador and
Dani, 1995; Galzi et al., 1996). For chicken homomeric 7
AChRs, it was shown that divalent cation binding sites in extracellular
domains are likely to mediate potentiation of the response by
extracellular Ca++. It was proposed that Ca++
potentiates responses by direct interaction with the nicotinic ligand
binding site of the AChRs. Substitution of 2 for 4 subunits virtually eliminates Ca++ potentiation of the human 3
AChR responses. This suggests that extracellular Ca++ can
modulate AChR function via "structural subunits" as
well. Considering that the ligand binding pocket is formed by the
interface of the and AChR subunits, a 2-located site of the
domain responsible for the Ca++ potentiation is not
unexpected. Differential Ca++ potentiation of the 32
and 34 AChRs could account for differences of Ca++
flux observed for these AChRs recombinantly expressed in HEK-293 cells
(Mahaffy et al., 1996).
Recombinant and native 3 AChRs.
As shown by Conroy and Berg
(1995) on neurons of chick ciliary ganglia, immunoprecipitation and
immunoblot analysis strongly suggests that at least a portion of 3
AChRs contain four kinds of subunits: 3, 2, 4 and 5.
Coexpression of the corresponding human subunits in Xenopus
oocytes resulted in functional AChRs with a distinct
concentration-response curve for ACh. Hill equation fit indicated at
least two populations of AChRs. One population (55%-65% of the
total) had significantly lower affinity for ACh (EC50 = 345 µM) and DMPP (EC50 = 110 µM) compared with the other subunit combinations tested (table 1), which suggests that it might
result from the combination of four kinds of subunits. The higher-affinity site had affinities for both ACh and DMPP close to the
values for 32 AChRs. The distribution of affinities for ACh
estimated for oocytes expressing all four subunits indicates that the
contribution of 325 AChRs to the mixture of AChRs expressed
was negligible. Immune precipitation analysis showed that greater than
70% of the 3 AChRs contained both 5 and 2 subunits. This
strongly suggests that the population of 3 AChRs with unusually low
affinity for ACh contains all four subunits. Overall, data on
immunoidentification confirm not only the high efficiency of coassembly
of 5 subunits with 3 and 2 or with 3 and 4 AChR subunits
as previously determined (Wang et al., 1996) but also
indicate the incorporation of 5 subunits into 3 AChRs containing
both 2 and 4 subunits.
42 AChRs are the dominant non-bungarotoxin binding neuronal AChR in the brain (Whiting and Lindstrom, 1986, Flores et al., 1992). A significant part (up to 25%) of human
neocortex 4-containing AChRs could be immunodepleted by
preadsorbtion with mAb 210, which binds to both 3 and 5 subunits.
The amount of mAb210-immunodepleted 4 containing AChRs appears to be
larger than the amount of AChRs that could be immunoisolated from
neocortex by mAb210 alone. This discrepancy might be in part due to
degradation of the AChRs during the day required for the additional
step of immunodepletion. A majority of the AChRs that bind to mAb210
could be depleted by the 4-specific mAb299. These data strongly
suggest that the 5 subunit is incorporated in some 42 AChRs,
although incorporation of 3 or of some other unknown AChR subunit
that has affinity for mAb210 could not be excluded. According to the in situ hybridization studies, expression of 3, that of
4 and that of 5 have different but overlapping patterns in
mammalian brain. Cerebral cortex contains messages for all of these
AChR subunits as well as for 2 subunits (Deneris et al.,
1991).
As expected, ganglionic-type neurons from the human neuroblastoma cell
line SH-SY5Y were found to have a significantly different pattern of
AChR subunit expression. 35 subunit-containing AChRs account for
all of the high-affinity [3H]epibatidine binding sites in
these cells, with no detectable expression of 4 subunits. Half of
these 35 AChRs contain 2 subunits. 4 subunits probably
substitute for 2 in the rest of the AChRs.
Comparison of the data on AChR subunit expression in the human
neocortex with the data obtained from the rat total brain extract reveals significant differences in levels of expression of mAb210 binding AChRs. A small but significant part of the 4-containing AChRs from the rat brain could be immunodepleted by binding to mAb210.
The overall level of 3 and 5 AChR subunits is very small (~4%)
relative to 2 and 4 subunits, a level much lower than in the
human neocortex. These differences could result from differences in the
origin of the brain tissue, with human cortex representing only its
local distribution of the AChRs. Alternatively, differences in
expression could be interpreted as due to differences between rats and
humans.
Unlike message for the 4 AChR subunits, which has a rather diffuse
and diverse pattern of expression in the vertebrate brain, the patterns
of 3 and 5 subunit expression are much more localized. 5
subunit mRNAs are present at modest levels in the cortex, at higher
levels in the interpeduncular nucleus and at the highest levels in the
ventral tegmental area and substantia nigra pars compacta (Wada
et al., 1989; Boulter et al., 1990). These areas include regions in which there is nicotinic facilitation of dopamine release. Recently it has been suggested (Le Novere and Changeux, 1995)
that some of the regions thought to contain 3 on the basis of the
in situ hybridization studies actually contain the closely related but pharmacologically distinct 6 subunit (Gerzanich et al., 1997). This prediction has been confirmed
immunohistochemically (Goldner et al., 1997).
Pharmacological, kinetic and Ca++-dependent effects of 5
subunits on 3 AChRs imply that 5 subunits could be utilized
effectively for fine-tuning neuronal nicotinic AChR function in
vivo. In the periphery, synaptic 3 AChRs from human autonomic
neurons are the most likely to be functionally affected by the presence
of 5 AChR subunits. In the human brain, 5 subunits may be
associated with a small fraction of 42 AChRs as well as with 3
AChRs.
| |
Footnotes |
|---|
Accepted for publication March 2, 1998.
Received for publication November 6, 1997.
1 Research in the laboratory of J.L. is supported by grants from the NIH (NS11323), the Smokeless Tobacco Research Council, Inc., The Council for Tobacco Research-USA, Inc., and the Muscular Dystrophy Association.
Send reprint requests to: Dr. Jon Lindstrom, 217 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104-6074.
| |
Abbreviations |
|---|
AChR, acetylcholine receptor; mAb, monoclonal antibody; DMPP, 1,1-dimethyl-4-phenylpiperazinium.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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|---|
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), nicotine (
), cytisine
(
) and DMPP (
) were built for the four combinations of the
subunits tested. Curves for ACh and nicotine are from Wang et
al. (1996)
