Phylogenetic conservation of disulfide-linked, dimeric acetylcholine receptor pentamers in southern ocean electric rays
1 School of Biochemistry & Molecular Biology, Australian National
University, Canberra, ACT 0200, Australia
2 John Curtin School of Medical Research, Australian National University,
Canberra, ACT 0200, Australia
3 MCD Biology, University of Colorado, Boulder, CO 80309, USA
* Author for correspondence (e-mail: louise.tierney{at}anu.edu.au)
Accepted 15 July 2004
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Summary |
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Key words: acetylcholine receptor, ligand-gated ion channel, electric fish, dimer, clustering, Torpedo macneilli, Torpedo marmorata, Hypnos monopterigium, Narcine tasmaniensis
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Introduction |
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The prototypal ligand-gated ion channel is the nicotinic acetylcholine
receptor. This receptor was the first neurotransmitter receptor identified as
a molecular entity (Noda et al.,
1982,
1983
) and the first to be
isolated and purified in an active form
(Miledi et al., 1971
). This
receptor was subsequently reconstituted in an artificial membrane system and
shown to retain its physiological properties in a quantitative fashion
(reviewed in Changeux et al.,
1983
; Conti-Tronconi and
Raftery, 1982
). It is also the only member of this family for
which we have any direct structural information
(Miyazawa et al., 1999
; Unwin,
1993
,
1995
). Research into its
structural and functional properties has benefited greatly from an abundant
source of this receptor in the specialized (muscle-derived) electric organ of
electric rays, where it is responsible for generating the electrical current
used to stun its prey.
The acetylcholine receptor, affinity purified from the electric ray
Torpedo californica, was shown by micro-sequence analysis to be a
pentamer composed of four homologous subunits with a stoichiometry of two
subunits and one each of the ß,
and
subunits
(
2ß
; Raftery et
al., 1980
). The density of the acetylcholine receptors is so high
in the electric organ that they appear as an almost crystalline array and this
allowed the use of electron microscopy to reveal the morphology of the
receptor. By negative staining, distinctive rosette-like structures were seen
densely covering the postsynaptic membrane isolated from the electric organ.
These same structures were observed with solubilized, purified acetylcholine
receptors, validating their identification as acetylcholine receptor molecules
(Chang et al., 1977
). In
contrast to neural and muscle forms of the acetylcholine receptor identified
subsequently, the native receptor derived from the electric organ of
specialized fish is a dimer, cross-linked by a disulfide bridge between
subunits in adjacent pentamers
(Hamilton et al., 1979
).
Early investigators obtained the acetylcholine receptor from the electric
rays Torpedo californica, Torpedo marmorata, Torpedo nobiliana and
Torpedo oscellata or occasionally from the lessser known species the
numbfish Narcine entemedor and Narcine braziliensis and the
electric eel Electrophorus electricus (reviewed in
Conti-Tronconi and Raftery,
1982). Indeed, it is the native receptor isolated from T.
marmorata that has been used in combination with cryo-electron microscopy
to derive direct structural information for this family of ligand-gated ion
channels (Miyazawa et al.,
1999
; Unwin, 1993
,
1995
). Although T.
marmorata has been used in such studies, its sequence data are
incomplete. We report here the nucleotide sequence of the three
uncharacterized subunits (ß,
and
).
To date, all attempts to solve the acetylcholine receptor's structure at
high resolution by X-ray diffraction have been unsuccessful, mainly due to the
low quality of the crystals. This is a problem common to crystallographic
studies of membrane proteins owing to the disorder caused by the necessity for
detergent-based solvents and the inherent mobility of transmembrane
-helices. A number of membrane protein structures have been solved
recently, many of which have employed the use of natural variation to select a
suitable natural variant for X-ray crystallography
(Chang and Roth, 2001
;
Chang et al., 1998
;
Doyle et al., 1998
;
Dutzler et al., 2002
;
Jiang et al., 2003
). We have
analyzed the neuromuscular acetylcholine receptor cDNAs and proteins sourced
from three southern ocean electric fish that may provide alternative sources
of protein for both biochemical and structural studies. The electric fish
investigated in this study are members of three different families and all are
endemic to Australian waters. The electric ray Torpedo macneilli is
in the same family as Torpedo californica and Torpedo
marmorata. The coffin ray, Hypnos monopterigium, is a member of
the Hypnidae family, although it is sometimes given subfamily status within
the family Torpedinidae. The numbfish Narcine tasmaniensis is the
smallest of the electric rays and is a member of the family Narcinidae. Our
results show that the acetylcholine receptor sourced from electric rays is
highly conserved across these different families, and alignment of their
deduced protein sequences with the equivalent human genes shows them to be a
good model system for studying the human acetylcholine receptor at the
neuromuscular junction. The large amount of sequence information available
today further enforces the homologous nature of the subunits for any receptor
in this family. Thus, in general, information obtained for one member is
applicable to all members of the ligand-gated ion channel family.
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Materials and methods |
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RT-PCR products were subcloned into the pGEM-T Easy vector (Promega,
Madison, WI, USA) and two or three isolates from each of the four
subunit-specific cDNA clones were sequenced on both the positive and negative
strands. Automated sequencing was performed using BigDye3 (ABI, Foster City,
CA, USA) sequencing mix and run on an ABI377 DNA Sequencer. Sequence data were
analyzed using MacVector software, and sequence alignments created in
ClustalW. All sequences have been deposited in GenBank (accession numbers:
betaAChRTmar AY472103; gammaAChRTmar AY472104; deltaAChRTmar AY472105;
alphaAChRHmon AY472106; betaAChRHmon AY472107; gammaAChRHmon AY472108;
deltaAChRHmon AY472109; alphaAChRNtas AY472110; deltaAChRNtas AY472111). The
T. marmorata subunit sequence has previously been published
(accession no. J00963; Noda et al.,
1982
) and its sequencing here serves as a positive control for the
RNA extraction and RT-PCR procedures.
Synthesis of the TDAC affinity ligand 4,7,10-trioxa-1-trityl,13-tridecanediamine (compound 1)
To 170 g of 4,7,10-trioxa-1,13-tridecanediamine (0.77 moles) dissolved in
500 ml of tetra-hydro furan (THF) was added 15.6 g of triethylamine (0.154
moles). To this solution was added 25 g (0.077 moles) of trityl bromide
dissolved in 250 ml of THF via a drop funnel over a 1 h period. The
solution was allowed to stir for 4 h and solvent removed in vacuo.
The remaining liquid was dissolved in EtOAc and washed with 3x500 ml of
water, and the organic phase was filtered through anhydrous magnesium sulfate
and dried in vacuo. The resultant product gave a single spot on thin
layer chromatography (TLC) at an Rf of 0.2 in 80:20 IPA:EtOAc. The yield
was 95%.
4,7,10-trioxa-1-trityl,13-carboxyethyl-2-bromide-tridecanediamine (compound 2)
Compound 1 (33.91 g/0.0733 moles) was dissolved in 300 ml of dry
Et2O and cooled to 0°C. 5.8 g (0.0733 moles) of pyridine was
added dropwise and then 13.6 g (0.0733 moles) of ClC(O)Et-Br was added
dropwise with stirring. After the completion of this addition, the solution
was allowed to stir for 1 h and warm to room temperature. The ether solution
was extracted with 500 ml of water and washed with an additional 3x500
ml of water. The organic layer was filtered through anhydrous magnesium
sulfate, and solvent was removed in vacuo. The product showed a
single spot by TLC and was used directly in the next step.
4,7,10-trioxa-1-trityl,13-carboxyethyl-2-trimethylamine-tridecanediamine (compound 3)
Compound 2 was dissolved in toluene (200 ml), and 10 ml of liquid
trimethylamine was bubbled through the solution via a cannular. The
reaction mixture immediately turned cloudy as a white precipitate appeared.
The reaction was allowed to stir overnight and solvent removed in
vacuo. The resultant syrup was dissolved in MeOH, a slurry was made with
silica gel and the excess MeOH was removed in vacuo. The silica
slurry was washed with EtOAc to remove all excess starting material and then
extracted with EtOH in a soxhlet extractor. Yield was 98% from compound 2.
4,7,10-trioxa-1,13-carboxyethyl-2-trimethylamine-tridecanediamine (TDAC)
The trityl group was removed by treatment with toluene sulfonic acid in
EtOH and monitoring with TLC. The product was extracted into water and washed
with CHCl3 to remove trityl-OH and then lyophilized to yield
essentially pure TDAC (Fig. 1).
Yield was 99%. The product showed a M2+ mass of 174.60 compared
with the expected 174.65. The lyophilized product was used directly in
coupling to N-hydroxysuccinimide-activated Sepharose fast flow
(NHS-FF; Amersham Pharmacia Biotech, Piscataway, NJ, USA), as per the
manufacturer's instructions, to produce the TDAC affinity column.
|
Purification of the acetylcholine receptor using a novel ligand affinity column
The acetylcholine receptor was purified from the electric organ of the
three southern ocean electric rays (T. macneilli, H. monopterigium
and N. tasmaniensis) and from T. marmorata from the North
Atlantic. Each tissue was treated identically for comparative purposes. Frozen
tissue (50 g) was thawed in 150 ml of ice-cold homogenization buffer (buffer
A: 20 mmol l1 Na phosphate, pH 7.4, 400 mmol
l1 NaCl). The tissue was homogenized in a blender (high
speed, 2 min), and insoluble material removed by centrifugation at 6000
g for 10 min. The supernatant was filtered through
cheese-cloth, 75 µl of a protease inhibitor cocktail was added (stock
solution: 1.0 mg ml1 leupeptin, 1.0 mg ml1
pepstatin, 50 mg ml1 PMSF) and then re-centrifuged to pellet
the membranes (150 000 g, Ti45 rotor, 30 min, 4°C).
Membrane pellets were weighed and resuspended in 10x vol./mass buffer B
(20 mmol l1 Na phosphate, pH 7.4, 80 mmol
l1 NaCl) plus protease inhibitors (1.0 µl
ml1). Triton X-100 was added to a final concentration of
1.5% and the solution left stirring on ice for 60 min. Solubilized protein was
recovered following centrifugation in a Ti45 rotor at 150 000
g for 30 min at 4°C, and following a twofold dilution with
water the supernatant was loaded directly onto the TDAC affinity column (4.0
ml). The chromatography was performed at 4°C on an Akta Explorer
Chromatography workstation (Amersham Pharmacia Biotech). Buffers for the
chromatography contained 0.2 mmol l1 Brij35 instead of
Triton X-100, enabling detection of protein by absorbance at 215 and 280 nm.
Bound receptor was eluted from the TDAC resin using an NaCl gradient from 0.04
mol l1 to 1.0 mol l1 over 15 column
volumes. Buffer A: 20 mmol l1 Na phosphate, pH 7.4, 0.2 mmol
l1 Brij35. Buffer B: 20 mmol l1 Na
phosphate, pH 7.4, 0.2 mmol l1 Brij35, 1.6 mol
l1 NaCl. Using a flow rate of 4.0 ml min1,
the chromatography step was completed within 80 min.
Protein eluted from the TDAC column was diluted fourfold in buffer containing 20 mmol l1 Na phosphate, pH 7.4, 0.2 mmol l1 Brij35 and applied to a 1.0 ml ResourceQ anion exchange column (Amersham Pharmacia Biotech). Protein was eluted from this column using a 0.041.0 mol l1 NaCl gradient over 40 column volumes generated with the same buffers used for the TDAC affinity chromatography and run at 4.0 ml min1. Gel permeation chromatography was performed on a Superdex200 column and run at 0.2 ml min1 in buffer containing 20 mmol l1 Na phosphate, pH 7.4, 0.2 mmol l1 Brij35, 80 mmol l1 NaCl. The system was calibrated using the gel permeation protein standard markers from BioRad (Hercules, CA, USA), which contain a mixture of proteins ranging in molecular mass from 1.35 to 670 kDa.
Equilibrium binding of 125I--bungarotoxin to detergent-purified acetylcholine receptors
Radio-ligand binding experiments were carried out essentially as described
previously (Schmidt and Raftery,
1973), using the rapid filtration method through anionic DE-81
filters to bind receptors and wash away unbound ligand. Purified receptor was
diluted into buffer containing 20 mmol l1 Na phosphate, pH
7.4, 80 mmol l1 NaCl, 1.5% Triton X-100 and used in binding
studies at a final concentration of 6.6 µg ml1. Each of
the four different receptor preparations was tested at eight toxin
concentrations ranging from 0.5 to 75.0 nmol l1
-bungarotoxin (
-Bgt). Unlabeled toxin was diluted to a final
concentration of 250 nmol l1 and spiked with
3-[125I]iodotyrosyl-
-bungarotoxin (Amersham Pharmacia
Biotech; specific activity 74 TBq mmol l1). Each
concentration was tested in triplicate and the assay repeated. To determine
the level of non-specific binding arising from the filters or the protein, no
protein was added or a 100-fold excess of cold
-Bgt was added,
respectively. No significant difference was seen in the level of counts
between these two experiments, indicating that, at the protein concentration
used, non-specific binding was due to the filters. Specific binding was
calculated by subtracting non-specific binding from the total binding for each
radio-ligand concentration tested. The data were fitted to the equation for a
single binding site by least square non-linear regression using the program
PrismTM (version 1.03; GraphPad Software, San Diego, CA, USA). The
equation used was:
Y=BmaxxX/(Kd+X), where
Bmax is the maximum binding, X is the concentration of
-Bgt and Kd is the dissociation constant.
Electron microscopy
Copper grids (TAAB, 400 mesh) on a formvar film were coated with a thin
layer of carbon and glow discharged for 12 min immediately prior to
use. Protein samples (25 µl) were added to the carbon-coated grids
and stained with 3% uranyl acetate. Samples were viewed using a Hitachi 7100
electron microscope operating at 75 kV. Specimens were viewed under the
electron microscope at 30 00040 000x magnification. Images of the
TDAC affinity-purified material were acquired using an SIS Megview III
Widefield CCD camera and saved as 16-bit images at 100 000150
000x magnification using the software analySIS (Soft Imaging Systems,
Munster, Germany).
Protein detection and quantification
Protein components were identified and their purity checked on sodium
dodecyl sulfatepolyacrylamide (SDSPAGE) gels. Samples were run
on NuPAGE 412% Bis-Tris polyacrylamide gels (Invitrogen, Carlsbad, CA,
USA) using MES (morphino-ethane sulfonic acid) as the buffer. Each sample was
diluted twofold in electrophoresis sample buffer (5% SDS, 500 mmol
l1 dithiothreitol, 6 mol l1 urea, 62 mmol
l1 Tris, pH 6.8, 10% glycerol, 0.002% bromophenol blue) and
left at 37°C for 2 h prior to electrophoresis. Gels were stained in
Coomassie brilliant blue (R350).
Protein concentrations were determined using the Pierce BCA protein assay (Pierce, Rockford, IL, USA) in accordance with the manufacturer's instructions and employed bovine serum albumin (BSA) as the standard.
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Results |
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Alignment of the northern hemisphere T. marmorata and southern hemisphere H. monopterigium and N. tasmaniensis acetylcholine receptor protein sequences with the sequences previously determined from the related electric ray T. californica and the equivalent human sequences was performed using the program ClustalW. Table 1 displays the resultant sequence identity scores. Across the fish species, each corresponding subunit is highly conserved (9499% identity). The only subunit that departs slightly from this trend is the ß subunit from H. monopterigium, in which the sequence identity falls to 88% compared with the equivalent subunit from T. californica. The identity between non-equivalent pairs of subunits from the electric fish sequences ranges from 32 to 46%.
|
The degree of sequence conservation between the non-equivalent subunit
pairs is similar when this comparison is made with the subunits from human
muscle; for example, T. marmorata and T. californica
, 33% identity; T. marmorata
and human
, 33%
identity. Comparison of the corresponding subunits from fish and humans
reveals a striking conservation of the
subunits (
78%), which is
significantly higher than that seen between the other pairs of equivalent
subunits (5358%). Finally, of note is the slightly higher sequence
identity observed between
and
subunits from the different
species (around 46% identity). This trend is seen in all species comparisons
and reflects their divergence, in evolutionary terms, from an intermediate
species (Ortells and Lunt,
1995
). It should be noted that many of the substitutions between
corresponding and between non-equivalent subunits are conservative, thus
tending to further relate the polypeptides.
The natural variation in the acetylcholine receptor observed between
species may provide information about structural and functional constraints
placed on the amino acid sequences of these receptors. In order to view the
distribution of amino acid changes, Table
2 groups these differences relative to T. californica
into those that lie in the extracellular or pore-forming domains of the
receptor. Using the atomic structure of the analogous acetylcholine binding
protein (Brejc et al., 2001) as
the framework, the amino acid differences have been grouped into those that
lie in the ß-strands and
-helices or the loops of the
extracellular domain. The distribution of those that lie in the transmembrane
-helices as opposed to the intervening loops of the pore-forming domain
is based on the latest structural information from cryo-electron microscopic
images of the acetylcholine receptor isolated from T. marmorata
(Miyazawa et al., 2003
). When
these data are viewed in this context, it is apparent that the amino acid
sequence differences are uniformly distributed between both the secondary
structure elements and between the two domains. As might be expected for
members of the same genus, the number of amino acid differences between
equivalent T. marmorata and T. californica proteins is low.
For example, there are just six differences in the
subunits and all
are conservative amino acid changes (except G254V) resulting from single
nucleotide changes (S66N, G254V, I315V, D342N, V347L, S448C). The greatest
number of differences observed with respect to T. californica is in
the ß subunit from H. monopterigium, which carries 51 amino acid
changes. Overall, the majority of amino acid differences observed between
T. californica and the newly sequenced electric ray subunits are
conservative in nature, including those that fall within the ligand-binding
loops and the transmembrane
-helices. On the other hand, of the
residues that are conserved, it is interesting to note that the penultimate
residue in the
subunit, a cysteine residue, is present in all electric
ray species sequenced to date.
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Affinity purification of functional, native acetylcholine receptors
The membrane-containing fraction isolated by differential sedimentation
from the fish electric organs was resuspended in buffer containing Triton
X-100, and the resulting solubilized protein was run through an affinity
column specific for intact acetylcholine receptors
(Fig. 3A). A salt gradient
(0.41.0 mol l1 NaCl) was used to elute bound
receptors from the affinity resin, and a single peak was observed. This peak
contained receptor that was 9095% pure as judged from Coomassie-stained
gels run under reducing conditions (Fig.
4). The assignment of protein bands corresponding to each of the
four acetylcholine subunits was confirmed using specific antibodies in western
blots (Tierney and Unwin,
2000; data not shown). As expected, the migration pattern of the
four subunits was identical to that determined previously for the
acetylcholine receptor subunits isolated from the torpedo ray (reviewed in
Conti-Tronconi and Raftery,
1982
). The protein band migrating between the
and ß
subunits most likely represents the
subunit, as it is often seen to
run as a doublet when purified from native sources. The only major
contaminating protein in the affinity-purified material of all the fish
preparations is most likely one of the subunits from a
Na+/K+-ATPase. This rationale was based on its
electrophoretic mobility (Fig.
4, migrating just below the 98 kDa marker) and its reported
contamination of all other preparations using the electric organ as its source
of receptor protein (e.g. Waser et al.,
1989
). This protein was removed effectively on a Q-sepharose anion
exchange resin, where it eluted later than the receptor, as shown in
Fig. 3B. The purified receptor
fraction ran as a single peak on a Superdex200 gel permeation column,
suggesting that the fraction is homogeneous
(Fig. 3C). Its elution, close
to the void volume of the column (670 kDa molecular mass marker), was
consistent with the native receptor being a dimer.
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|
The receptors isolated from each of the four fish species behaved
identically on the chromatography system, with the only difference being in
the amount of protein purified. Starting with the same amount of electric
organ (i.e. 50 g), the largest quantity of purified receptor was isolated from
T. marmorata (6.9 mg) followed by T. macneilli (4.0 mg) and
H. monopterigium (4.0 mg); interestingly, the lowest yield was from
the smallest specimen, N. tasmaniensis (3.0 mg). The binding capacity
of the resin was not exceeded in any of the purification runs as judged by the
lack of immunoreactive protein in the unbound and wash fractions when examined
by western blotting using antisera specific for the subunit (data not
shown).
Equilibrium binding of purified acetylcholine receptors to 125I--bungarotoxin
Although the acetylcholine receptor was purified on an agonist affinity
resin, the binding properties of the detergent-purified receptors were tested
further in -bungarotoxin equilibrium binding assays in order to obtain
dissociation constants for comparison with alternative purification procedures
and with receptors from different species.
Fig. 5 shows the results of a
typical binding experiment in which the line represents the best fit of the
data to the equation for a single binding site. The detergent-solubilized and
affinity-purified acetylcholine receptors all exhibited values for
Kd in the nanomolar range for the snake neurotoxin. The
dissociation constants derived from the data for the individual species were
not significantly different from each other
(Table 3). Dissociation
constants for
-bungarotoxin in the low nanomolar range have been
reported previously for T. californica receptors assayed in
situ from membrane preparations (Chang
et al., 1977
; Vandlen et al.,
1976
), detergent-purified preparations
(Waser et al., 1989
) and
recombinantly expressed acetylcholine receptors
(Sine, 1997
).
|
|
Electron microscopy of acetylcholine receptors purified from fish electric organs
The detergent-solubilized and affinity-purified acetylcholine receptors
from the electric rays were viewed in negative stain under the electron
microscope. All exhibited the distinctive rosette-like structure with a
central stain-filled pore that is typical of ligand-gated ion channels
(Brisson and Unwin, 1985).
Furthermore, the acetylcholine receptors isolated not only from T.
marmorata but also those isolated from all three southern ocean fish
species remained as dimers when purified under these conditions
(Fig. 6).
|
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Discussion |
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A cholinergic affinity resin was used to purify intact acetylcholine
receptors following their solubilization from the membrane fraction of the
fish's electric organ. The resin constitutes a chemically modified
carbamylcholine molecule, in which the ester has been substituted by an amide,
thus eliminating the problem associated with hydrolysis of this ester bond.
Early attempts to affinity purify the receptor using carbamylcholine as the
immobilized ligand were hampered by this problem
(Vandlen et al., 1976). The
hydrolysis of the carbamylcholine off this resin was attributed to the actions
of acetylcholine esterase, which is known to hydrolyse carbamylcholine and is
present in significant amounts in the detergent extracts from electric fishes.
The interaction between the immobilised TDAC ligand and the acetylcholine
receptor, while being specific, is relatively easy to disrupt with an
increasing NaCl gradient (160320 mmol l1 NaCl;
Fig. 3A). It has long been
established that the binding of ligands to the acetylcholine receptor is
sensitive to the presence of cations (Akk
and Auerbach, 1996
; Schmidt
and Raftery, 1974
). Therefore, it is perhaps not surprising that
the receptor's interaction with the immobilised TDAC ligand is sensitive to
the NaCl concentration. Indeed, the NaCl concentration must be optimised in
order to ensure that the TDAC resin acts as an affinity column and not as a
general anion exchange column.
Affinity chromatography of the acetylcholine receptor has traditionally
employed either reversibly binding, acetylcholine analogues or the snake venom
-toxins immobilized to an inert support (e.g.
Chang et al., 1977
;
Sobel and Changeux, 1977
;
Vandlen et al., 1976
;
Waser et al., 1989
). However,
the purification of the acetylcholine receptor proteins has been incomplete,
the recovery of the receptors from the affinity columns was not satisfactory
or the affinity matrices were hydrolyzable. Previous purification procedures
have also encountered problems associated with proteolysis of the subunits and
with the reduction of the inter-pentamer disulfide bond that leads to the
presence of significant amounts of monomeric receptor. Both of these problems
were exacerbated in many of the reports because of the length of time it took
to perform the chromatography under manual conditions. An important advantage
of our procedure is its automation, which in turn minimizes the time and
controls the temperature during chromatography. Proteolysis was not evident in
any of our receptor preparations as judged from western blotting. Furthermore,
dimeric receptors were isolated under the conditions used as illustrated by
electron microscopy and implied by the homogeneity of the purified receptor
run on gel permeation chromatography. The acetylcholine receptors purified to
date do not readily form crystals that diffract at high resolution. Native
acetylcholine receptors obtained from southern ocean electric rays and rapidly
purified on the TDAC affinity resin offer alternative sources from which to
attempt crystallization trials.
The formation of the receptor dimer in T. californica is known to
be the result of a disulfide bond forming between the penultimate cysteine
residue of subunits in adjacent pentamers
(Hamilton et al., 1979
). This
cysteine residue is conserved in all species examined here and it would appear
that this mechanism of receptor pairing is present also in the southern ocean
electric rays, suggesting that dimerization through a cysteine residue is a
property of all acetylcholine receptors found in the electric organs of
specialized fish. Functioning of the nicotinic synapse requires not only the
intrinsic ion-gating properties of the acetylcholine receptors but also a
critical density of acetylcholine receptor molecules (reviewed in
Sanes and Lichtman, 2001
).
Cytoskeletal anchoring and clustering of receptor molecules in the plasma
membrane occurs for all members of the ligand-gated ion channel family
(reviewed in Colledge and Froehner,
1998
). Their specialized organization and concentration are
initiated through interactions with receptor-specific cytoplasmic proteins
such as rapsyn, gephyrin and GABAA receptor associating protein
(GABARAP). It is the interaction of these proteins with microtubules that
anchors receptors at specific sites in the membrane. In addition to these
constraining mechanisms, limiting the lateral diffusion of ligand-gated ion
channels in the membrane, there is increasing evidence of a physical
interaction between individual receptors. The clustering that occurs
via such proteinprotein interactions has been shown to occur
between intracellular domains of GABAA receptor pentamers and has
effects on both channel kinetics (Chen et
al., 2000
) and channel conductance
(Everitt et al., 2004
). The
physiological role of the unique, additional clustering mechanism of
acetylcholine receptors in the electric organ via an inter-pentamer
disulfide bond remains to be elucidated. It may act, however, to stabilize
receptor clusters, thereby facilitating synchronous gating of the ion
channels, and provide a physical mechanism for regulating information flow by
enhancing receptorreceptor cross-talk.
Phylogenetically, the acetylcholine receptor is highly conserved across species, whether it be stacked in the electric organs of fish or clustered at the neuromuscular junctions of vertebrates, including humans. The sequencing and functional characterization of the abundant acetylcholine receptors from both southern and northern hemisphere electric rays described here shows them to be a good model system for the pharmacologically important human receptor found at the neuromuscular junction.
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Acknowledgments |
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References |
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