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