BIOCHEMICAL
Vol. 68, No. 2, 1976
AND BIOPHYSICAL PFSEARCH COMMUNICATIONS
CHARACTERIZATION OF A NICOTINIC ACETYLCHOLINE RECEPTOR FROM RABBIT MUSCLE AND RECONSTITUTION IN PLANAR PHOSPHOLIPID BILAYERS R. J. Bradley Neurosciences Alabama 35294
Received
SKELETAL
, J. H. Howell , W. 0. Romine , G. F. Carl , and G. E. Kemp Program, The University of Alabama in Birmingham, Birmingham, and New York State Department of Health, Roswell Park Memorial 14263 Institute, Buffalo, New York
November
14,1975
acetylcholine receptor from rabbit skeletal muscle was isoSUMMARY. The nicotinic lated by affinity chromatography and characterized by 1251- 4 -Bungarotoxin binding Quanta1 conductance events were observed when and gel filtration chromatography. this material was added to planar phospholipid bilayers. These changes were stimulated by carbamylcholine and antagonized by curare, Butx, dithiothreitol and concanavalin A. The receptor preparation was found to bind 0.2 nMoles 1251- o( -Bungarotoxin per mg protein and the molecular weight was estimated to be 390,000. INTRODUCTION. isolated
Studies
nicotinic
By measuring that
the
lo-l1
x
conductance
noise
at the
range,
in voltage
in black
lipid
by affinity
chromatography
added
cholinergic
agents
reconstitution diaphragm induced lated
increases
(8)
have
containing
shown that
In fact,
has also
been reported,
after
the purification
(5).
extraction
membrane
events
normal filtration.
choline
bilayers
sealed
vesicles
reconstitution
dissolution
of Torpedo
to
suggested
reported
the
and denervated This that
membranes
material
were
stimu-
and curare.
transport
in vesicles
AChR by affinity
this
was sensitive
from Electrophorus
of Torpedo
Fourier
within
which (6)
(Butx)
which
have
from mammalian
membrane
and gel
fragments
mho.
formation
Kemp --et al.
from both
(1)
an estimate
AChR isolated
by O(-Bungarotoxin
successful the
with
10-l'
the
channel.
and Miledi
about
channel
conductance
in egg phosphatidyl
the AChR, form
agonist.
and after
X-100
ion
out if
ion
has yielded
reconstituted
multiple
but antagonized
to added
(8)
This
is
endplates
doped
obtained
by acetylcholine
endplate
discrete
of an AChR preparation
conductance
Torpedo
Katz
aggregation
by 1.5% Triton
colleagues
muscle
channel
muscle
and his
(3, 4).
to find
a functional
membranes
and exhibited
or tetrameric
laboratories
(AChR) contains
clamped
We have measured
brain
an orthogonal
frog
several
of the open AChR channel
current
mho (2).
in
receptor
conductance
of membrane
of 3.2
been underway
acetylcholine
electronic
predicted analysis
have
Changeux
(7)
and
Na+ in response of native
lipid
in sodium
chromatography
(9).
cholate
BIOCHEMICAL
Vol. 68, No. 2, 1976
We now report reconstitution MATERIALS
the
in black
purification lipid
AND BIOPHYSICAL
of AChR from rabbit
RESEARCH COMMUNICATIONS
skeletal
muscle
and its
membranes.
AND METHODS.
Receptor Extraction. Naja naja siamensis &cobrotoxin and a-bungarotoxin The preparation of the affinity gel using were obtained from the Miami Serpentarium. Affi-Gel (Bio-Rad Laboratories) has been described previously (5). Alternatively, pure d-cobrotoxin (0.25 mg/g gel) was bound to cyanogen bromide activated Sepharose Rabbit hindlimb muscle was placed in 0.5 M KCl, 50 mM 4B obtained from Pharmacia. The tissue was chopped in a Waring blender and sodium phosphate buffer, pH 7.4. The homogenate was centrifuged at homogenized in a motor driven glass homogenizer. 10,000 x g for 60 minutes and the pellet obtained was resuspended in 20 mM sodium This was then phosphate, pH 7.4 containing 0.4% Triton X-100 and rehomogenized. centrifuged at 13,000 x g for 60 minutes and the supernatant was applied to a neurotoxin affinity column as previously described (5). Some extractions utilized 0.4 mM phenylmethanesulfonyl fluoride as a protease inhibitor. The column was washed with X-100 and AChR was eluted with 1 M NaCl, 50 mM sodium phosphate, pH 7.4, 0.1% Triton 50 mM sodium phosphate, pH 7.4, 0.1% Triton X-100 and 1 M carbamylcholine chloride. The receptor was then dialyzed exhaustively against 0.1% Triton X-100, 50 mM sodium phosphate, pH 7.4. All procedures were carried out at 4'C. Toxin Binding and Molecular Weight Determination. Butx was radioiodinated w~ih Iodine-125, by the solid-state lactoperoxidase method of David and Reisfeld (lo), using 1 mg Butx (0.13 FM), 0.13 p.M KI, 0.3 mC Na 1251, 0.2 IAM H202 and 50 pg of Sepharose 4B-coupled lactoperoxidase. The reaction was stopped with sodium azide and the iodinated toxin was immediately loaded on a Sephadex CM-50 column (35 x 1.0 cm) equilibrated with 50 mM aaunonium acetate, pH 5.8. The labeled toxin was eluted in a linear gradient to a final buffer concentration of 300 mM ammonium acetate, pH 7.0. The toxin eluted as a single peak and was pooled, lyophilized and stored in aliquots as an approximately 5 PM solution in 5 mM sodium phosphate, pH 7. The toxin had a specific activity of 1400 C/mole. Routine assay for receptor was based on incubation with 13 nM 1251-Butx for 60 minutes at room temperature and elution of the receptor-toxin complex from 2 cm x 0.5 cm CM-50 Sephadex columns in 0.1% deoxycholate-10 mM NaP04, pH 7.4. Extensively dialyzed affinity column effluent was incubated with 0.2 n)Moles of I-Butx for 60 minutes at room temperature, the incubate was applied to a Sepharose 6B column (1.7 x 86 cm) and the sample was eluted in buffer containing 0.10% sodium deoxycholate. Five marker proteins of known molecular weight were chromatographed on the samT2Folumn under identical conditions and a molecular weight estimate for the AChR: I-Butx complex was determined by interpolation on a plot of log molecular weight vs. Ve/Vo. Protein was determined by the method of Lowry --et al. (11) using bovine serum albumin as a standard. 125
Bilayer Analysis. Lipid bilayers were formed using the methods first reported by Mueller (12) Soybean lipid Type 11-S from the Sigma Chemical Company was -et --al dissolved in n-decane 30 mg/ml and then passed through columns of basic and acidic alumina prior to use. The bilayer was spread across a 1 mm aperature in a polyethylene chamber using a hollow polyethylene spreader filled with lipid as previously described The aqueous phase consisted of 0.1 M NaCl. Calomel electrodes were used to (5). apply a potential across the membrane and to measure the resulting current. A battery delivered 80 mV to the bilayer and the current generated were fed into a Keithley 427 amplifier. After the optically black configuration was achieved membranes were monitored for at least 60 minutes to insure baseline high resistance ( lo-' mho cm2). Samples of AChR up to 30 ~1 in buffer and 0.1% Triton X-100 were injected into the cis bilayer bath containing 5 ml of 0.1 M NaCl. For the investigation of pharmaco-
578
Vol.68,No.
BIOCHEMICAL
2, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
logical activity in the bilayer, all drugs used were injected on both sides of the membrane to produce the final concentration of dithiothreitol (DTT) receptor samples were first incubated presence of the stated concentration. RESULTS.
The use of cobrotoxin
to the purification affinity
as an affinity
of AChR electric
column
effluent
was labeled
6B column
that
had been
in Figure
1 results
calibrated
fish
(13)
with
125 I-Butx
with
in an estimated
ligand
the bathing media desired. In the case for 6 hours in the
has been successfully
and mammalian
marker
molecular
into
and gel
proteins. weight
diaphragm
applied
(14).
Dialyzed
filtered
on a Sepharose
Interpolation
in the
of 390,000
for
plot
the A&R:
Butx
‘L Figure
complex, of the
1.
A standard curve for estimation of molecular weight b el chromatography on a Sepharose 6B column (86 x 1.7 cm--f=!. The samples were eluted at 4°C in a buffer compound of 100 mM iiaC1; 2 mM histidine; 2 mM TES; 0.10% sodium deoxycholate, pH 7.4 at a flow rate of 5 ml/hr and 2 ml fractions were collected.
in excellent fractions
eluted
and toxin
binding
The later
fractionsof
agreement
with
from
the affinity
was found this
only
values
were
toxin
binding.
AChR purified
by gel
toxin
binding.
Shortly
the dialyzed
chamber in
the
thinned
in both
steady-state bilayers.
the
conductance There
amount
The best
preparations
lost
ductance
changes
composed
variation
was totally
absent
fractions
completely
filtration
less
induced than
of quanta1 (Figure
2).
579
bilayer
with
full
to the
variability
not demonstrate activity inner
between of activity at 4'C.
sizes
in conductance
and
bilayer
was only
in one month of several
activity
carbamylcholine.
bilayer
Activity
and the retention
The rise
An analysis
and did
was added
to increase.
events
both
eluted
retained
10% activity
(15).
that
inactive
eluate
began
elsewhere
showed
was some quantitative
of conductance
were
column
in the early
elution
after
reported
observed
preparations upon
storage.
These
con-
and continuous was a linear
Vol.68,No.
2, 1976
BIOCHEMICAL
2.6 -
AND BIOPHYSICAL
ST-Chymotrypsinogen
2.4 -
RESEARCH COMMUNICATIONS
A
\
2.2 Hen
Egg Albumin
l
\
9
2.0 -
. >”
IBRabbit
Muscle
1.6 -
1.4 -
I.21
I04
2
3
4
5
6769105
2
MOLECULAR
Figure
function
2.
When carbachol
ficantly
increase
bachol.
Carbachol were
Low concentrations after
effects
only
slightly
the AChR extracted
active
to the receptor.
probably
toxin
Curare
6 769106
has terminal
mannose
findings
conductance
the active.
intact
agents partially
from
Meunier
it
effect
Electrophorus
Therefore
had
where
--et al.
580
blocked
amounts little
on conductance (16)
the
have
and inhibits
residues.
of con-
of car-
(5 pl)of
effect
is a moderate
the AChR may contain
or N-acetylglucosamine
signi-
on the rate
in the presence
of small
Atropine
endplate
were
development.
and Butx
activity
mho
of Kemp --et al.
fluctuations
of 30 ~1 AChR material
of the AChR.
precipitates
the
pharmacological
of Con A had an antagonistic addition
with
of conductance
bilayer.
enhanced
to the
the
of several
the addition
markedly
in contrast
by rate
in the
after
otherwise
preparation
measured
the
development
ductance
which
5
WEIGHT
in agreement
to the bath,
as measured
1 describes
conductance
of AChR added,
was added
increased
Table
4
Continuous recording of a bilayer doped with muscle --.-AChR. Ordinate 10-Y mho, abscissa 30 set Rise time 100 msec - Keithley 427 current amplifier Applied voltage 80 mV The quanta1 events recorded here vary in size from 0.35 x 10-l' up to 1.3 x 10-g mho. Bathing solution 1M NaCl.
of the amount
(6).
3
material
on this antagonist. changes
shown that the binding a glycoprotein However,
Con A of radio which as in the
Vol.68,No.2,1976
Table
1.
BIOCHEMICAL
The effects of carbamylcholine conductance. Rate is measured addition of receptor preparation C is 30 ~1 of a fraction'later AChR is present.
~1 of muscle AChR added to bilayer bath in the presence of 5 x 1O-5 M carbamylcholine
:i 5 5 C
case of the other Con A blocks
drugs
after
activity
significantly.
conductance
these
polymers
sensitivity
There
and thus
reduce
(18).
The noise
after
on a disulphide
the bilayer
and its
linkage possible
bonds
(17).
which
electroplax
of multiple
indicating
that
(19).
The further
reversal
after
581
of a
be to break
down
as we have,
site
ACh application
of
is
conformation of this
in fact,
DTT decreases
the anionic
channel
investigation
the
by multiples
shown that
near
by 5,5'-dithiobis-(Z-nitrobenzoic
may be of some interest.
may reflect
events
has been
the active
reduced
the AChR from electroplax
of DTT might
endplate
receptor
DTT also
Our data
bond situated
at the frog
with
are manifested
AChR it
to say whether
or alters
that
One of the actions
a disulphide
recorded
DTT treatment
evidence
~~8.4 k7.4 k7.3 +l.l k4.5 +3.8 k9.4 k3.3 k10.8 kO.4
yet possible
the bilayer
the probability
In the
not
102.2 33.0 45.8 4.0 9.1 61.7 52.3 8.7 81.8 1.2
of the material
or polymers
or values.
is
into
of disulphide
the bilayer.
it
is biochemical
aggregates
value
tested,
Incubation
to ACh by reducing
decreased depend
incorporation.
of receptor
in
we have
of channels
by the oxidation
basic
binding
which
the incorporation
function
observed
Rate of Conductance Increase lo-lomho min-1 (no. of replications)
None 1 x 10s4M d-tubocurarine 8 x lo-% a-bungarotoxin (Butx) 6 x 10W4M dithiothreitol (DTT) 2 pg/ml concanavalin A (Con A) None 1 x 10W3M atropine None 2.5 x 10m4kl carbamylcholine None
ii 30 30
existence
and various ligands on the induced over the first ten minutes after - expressed as mean ?S.E.M. in the'elution profile where no
Drug Added
30
polymerizes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
also
may
phenomenon acid)
in
(DTNB)
Vol. 68, No. 2, 1976
Figure
2 is
material
a representative
to the bilayer.
these,
especially
frequent
size
events
event
recording
smaller is
that
bi-ionic
binding
measuring
Na+/Cl-
selectivity
of approximately
is
that
ductance
states
reported
for
the
isolated
arrays
conductance,
considerable
it
from electric
event.
obtained
that
this
The most
with
other
A similar
fundamen-
from mammalian
bilayer
activity
between
brain
showed
the progression
known polymerization
(20).
In support
The role
(5).
an
played
of con-
or aggregation
of this
junction
is (21)
properties
phenomenon
the observation
that
show orthogonal
or
by AChR aggregation
and cooperativity
of success
has recently
fish,
Reconstitution
its
of the from
other
phospholipid
information to judge
skeletal
muscle
tunately
not
would
appear
only
because
is
in modulating
of the receptor
may be of
by the authors have been
criticism
material possible
on vesicles
whether
because analogous
to be as successful of the greater
were
as that
flexibility
has not
conspicuous
which
is
the nature the receptor
receptor
to determine
work
the AChR in milligram
has proved
by the authors'
serious
whether
to purify
reconstitution
receptor
regarding
presently
and had an action
fish
(9),
A more
possible
functional
reconstitution,
vesicles
preparations.
been possible
laboratories
of functional
possible
off".
basic
fractions
and some of
importance.
Although
limited
and "turn
of this
the neuromuscular
densitization,
present
mho in 1 M NaCl,
some relationship
receptor
of particles.
on"
are
of receptor
3:1 at pH 7.4.
and the
from
the addition
x lo-l1
showed
is
here
membranes
rectangular
report
there
observed
freeze-fractured
the
potentials
after
"turn
3.5
RESEARCH COMMUNICATIONS
of events
rapidly
to be multiples
in neurotoxin
possible
sizes
events,
Studies
It
obtained
approximately
appear
was found
AND BIOPHYSICAL
Many different
the
channel
quanta1 tal
BIOCHEMICAL
involved
of the
into large
to that
of iostope
from
successful
in
only
technique
reported
for
of the planar
such properties
582
change
vesicles
as agonist
here, the fish bilayer
The only
some
provides that
does --in vivo.
amounts
reported
and reports
efflux
as it
incorporated
successful, absence.
the vesicle
responding
problem.
in their
of the conductance is
to be a difficult been
own admission that
quantities
(which
of material
it
such
is If is
not the
unfor-
required)
the reconstitution receptor
(9).
technique
that
independent
It it
conductance,
is is
Vol. 68, No. 2, 1976
channel
magnitude
Although fish
(9),
the AChR used
as gel drug
modification
can result
in alterations
behavior.
It
restoration neuromuscular
is ultimately of conductance
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
are
in this
inappropriate
work
of the bilayer
is
strongly
less
argues
the
than
that
behavior
that
some experimental
artifact.
that
as well
reported
electric
are
demonindeed
of the result
found
conductance in the
in the post-synaptic
junction.
ACKNOWLEDGEMENTS. 1 F22 NS02089-02
G. F. Carl
is a recipient
and W. 0. Romine
by USPHS grant
of a postdoctoral
is a NSF predoctoral
fellow.
fellowship This
work
was
NS 11356.
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Katz, B. and Miledi, R. (1972) J. Physiol., 224, 665-699. Anderson, C. R. and Stevens, C. F. (1973) J. Physiol., 235, 665-691. Romine, W. O., Goodall, M. 6. and Bradley, R. J. (1974) Fed. Proc., 33, 1253. Goodall, M. C., Bradley, R.J., Saccomani, G. and Romine, W. 0. (1974) Nature, 250, 68-70. Romine, W. O., Goodall, M. C., Peterson, J. and Bradley, R. J. (1974) Biochim. Biophys. Acta, 367, 316-325. J. 0. and Barnard, E. A. (1973) Biochem. Biophys. Res. Kemp, G., Dolly, Cormnun., 54, 607-613. Kasai, M. and Changeux, J. P. (1971) J. Memb. Biol., 6, l-23. Hazelbauer, G. and Changeux, J. P. (1974) Proc. Nat. Acad. Sci. U.S.A., 71, 1479-1483. Michaelson, D. M. and Raftery, M. A. (1974) Proc. Nat. Acad. Sci. U.S.A., 71, 4768-4772. David, G. S. and Reisfeld, R. A. (1974) Biochemistry, 13, 1014-1021. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265-275. Mueller, P., Rudin, D. O., Tien, H. T. and Wescott, W. C. (1963) J. Phys. Chem., 67, 534-535. E. and Widlund, L. (1972) FEBS Lett., 28, 107-111. Karlsson, E., Heilbronn,
583
a
evidence
and reconstitution
will those
to its
Preliminary
isolation
such investigations with
for
changes
as the magnitude
consistent
receptor.
in addition
conductance
of receptor
intact
by such different
the
in the nature
properties
for
chromatography,
of the conditions
hoped
pure
conductance
and affinity
of the AChR and are not that
supported
etc.
filtration
specificity,
consequence suggests
and lifetime,
the retention
techniques strated
BIOCHEMICAL
Vol. 68, No. 2, 1976
14. 15. 16. 17. 18. 19. 20. 21.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Brockes, J. P. and Hall, Z. W. (1975) Biochemistry, 14, 2092-2099. Hucho, F. and Changeux, J. P. (1973) FEBS Lett., 38, 11-15. Meunier, J. C., Sealock, R., Olsen, R. and Changeux, J. P. (1974) Eur. J. Biochem., 45, 371-394. Chang, H. W. (1974) Proc. Nat. Acad. Sci. U.S.A., 71, 2113-2117. Karlin, A. (1973) Fed. Proc., 32, 1847-1853. Landau, E. M. and Ben-Haim, D. (1974) Science, 185, 944-946. Edelstein, S. J., Beyer, W. B., Eldefrawi, A. T. and Eldefrawi, M. H. (1974) J. Biol. Chem., 250, 6101-6106. Heuser, J. E., Reese, T. S. and Landis, M. D. (1974) J. Neurocytol., 3, 108-131.
584