choline allosteric site in guanine nucleotide regulation of [3H]-L-QNB binding to muscarinic acetylcholine receptors

Vol. 113, No. 1, 1983 May 31, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 280-285

OBLIGATORY ROLE OF A TRIS/CHOLINE ALLOSTERIC SITE IN GUANINE NUCLEOTIDE REGULATION OF [3H]-L-QNB BINDING TO MUSCARINIC ACETYLCHOLINE RECEPTORS Kenneth

M.M. Murphy

and Antonio

Sas.tre*

Departments of Neuroscience and Physiology Johns Hopkins University School of Medicine 725 North Wolfe Street Baltimore, Maryland 21205 Received

March

21,

1983

Tris and choline reduce the maximal binding capacity (RT) of the mu carinic cholinergic antagonist [3H]-L-quinuclidinyl benzilate ([ s HI-L-QNB) to atria1 membranes, when compared to control values in physiological salt solution (PBS) or NaPi buffer. Addition of guanine nucleotides (GN) to incubations containing choline or Tris reverses the effect of choline and Tris on RT and restores it to levels determined in NaPi or GN addition fails to alter RT or KD values determined in PBS alone. NaPi or PBS in the absence of choline and Tris. This GN effect follows a nucleotide specificity similar to that of the GN regulatory proteins coupled Tris or choline are required for the expression of GN to adenylate cyclase. regulation of [3H]-L-QNB binding to muscarinic acetylcholine receptors An allosteric site recognizing choline and Tris appears involved in (mAChR). the interaction between the guanine nucleotide regulatory protein and antagonist binding to mAChR.

Guanine agonist

nucleotides

binding

Two or three isotherms

to the myocardial agonist

obtained

non-hydrolizable to the

mAChR, long group

sites

are

in the

form

we and others regarded

muscarinic necessary

absence

GTP analog

low affinity

Recently

(GN) and Na+ have well

Gpp(NH)p,

to regulation

*To whom all

correspondence

to account

the

effects

acetylcholine

of GN (4,5);

receptor

for

the

complex

in the presence

agonist

(1,2,3)

sites

have suggested

by ionic should

that

population, strength,

the

binding

of the

predominantly

antagonist are

sites

in fact

GN, choline

convert

in the

a heterogeneous

and Tris,

and

be addressed.

mAChR, muscarinic acetylcholine receptor; QNB, quinuclidinyl Abbreviations: benzilate; PBS, physiologic buffer solution; Gpp(NH)p, 5'guanylimidodiphosphate; GN, guanine nucleotides; RT, maximal binding capacity. 0006-291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

280

on

(mAChR).

(1,2,3).

as a homogeneous

subject

documented

8lOCHEMlCAL

Vol. 113, No. 1, 1983

monovalent

ions

et a . (7)

.

observations

proposed

Their

of sites

for

an d a decreased

antagonists

The interconversion

to adenylate

of this

effect

was not

Here we report

affinity

that

GN regulation

solutions,

an increased

agonists

in the

by a guanine the

of antagonist

is not

or exogenous

follows

nucleotide

displays

although

of a previously

such regulation of Tris

(9),

some of

reciprocal

for

mediated

cyclase

to integrate

affinity

presence

of GN.

nucleotide

regulatory

nucleotide

specificity

defined.

the occupation

(8)s and that

guanine

and have not

a GN-mediated

the receptor

was presumably

linked

Tris

are complex

model

postulated

in which

protein

the absence

interactions

an intriguing

model

interconversion

requires

RESEARCH COMMUNICATIONS

elucidated.

Burgisser these

These multiple

(6,7,8).

been completely

AND BIOPHYSICAL

choline.

This

effect,

linked

to adenylate

Materials

site

buffers

present

similar

mAChR

regulatory

in physiologic

specificity

proteins

to the

choline/Tris

demonstrable

a nucleotide

regulatory

defined

binding

in

in choline

to that

or

of the

cyclase

(9).

and Methods

Hearts were excised from mongrel dogs, CD rats, CD-l mice and Rana pipiens frogs and prepared as described (8). The homogenate was wire meshmiltered, centrifuged and 50 ~1 of the resuspended pellet was added to a final volume of 1 ml containing [3H]-L-QNE, drugs, nucleotides and buffer. Buffers used included a physiologic salt solution (PBS) (Dulbecco's phosphate-buffered saline, containing in mM: NaCl, 138; KCl, 2.6; CaC12, 1.0; MgS04, 0.5; Na2HPD4, 8; and KH2P04, 1.5), 50 mN NaPi or 50 mM Tris-HCl, all at pH 7.4 at 25'C. Incubations, filtrations and counting were carried out as Adenine and guanine nucleotides were obtained as Na previously described (8). salts from Sigma. Results As previously [3H]-L-QNB Tris

to myocardial

reveals

to parallel Addition

described

(8), tissue

a significantly experiments

carried reduced

carried

of GN restores

the

out

reduced

determined

in PBS and NaPi.

determined

in PBS or NaPi buffer.

of 213 L 10 fmol/mg exogenous

GN exhibited

Scatchard

protein.

analysis out

in the presence

maximal

binding

isotherms of choline

capacity

(RT)

(Figure

1).

RT determined

to the

levels

failed

Membranes

Membranes

281

to alter

assayed

assayed

apparent

in Tris

RT (68O/0

or

RT values

in NaPi displayed

in Tris

of

compared

in PBS or NaPi buffer

GN additions

a reduced

of binding

in the

of control),

absence which

an RT of was

Vol. 113, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

[3~]~~~ Figure

to control

concentration

Tris compared

values

in the

of GN increased

1 and Table caused

presence

the

a 3-fold

increase determined

approaching

in NaPi

in the K,, for

[3~]-~-~~~

for

of similar

ionic

1).

Gpp(NH)p

that

measured Table

Effects Atria1

This

RT determined

in NaPi buffer

52 + 10 pM; Table

to a value

of

of 100 UM Gpp(NH)p. by only

of Gpp(NH)p on Affinity Homogenates for [3H]-L-QNB

at lo-100 in NaPi,

the mAchR

not

and Maximal Assayed in

RT ('I'

control)

52 -+ 10

100

NaPi

+ Gpp(NH)p

46+

7

109 + 3

153 + 20

68 + 3

81 + 12

111 +- 5

KB values from

are

Scatchard

the

mean

z S.E.M.

The

concentration

presented

analysis

as mean of

of

the

of

Gpp(NH)p

+ S.E.M.

binding

percent

isotherms. of

was

control

100

PM.

282

affecting

Binding Capacity NaPi and Tris Buffer

(control)

+ tipp(NH)p

the KD

1

NaPi

Tris

strength

uM increased while

Ko (PM)

Tris

9"/e

1).

to KD values

(153 + 20 vs. in Tris

(fmol/mg)

1 Scatchard plot of [3H]-L-QNB binding to canine atria1 membranes in NaPi buffer without Gpp(NH)p (o), or with 100 uM Gpp(NH)p (0); in Tris buffer without Gpp(NH)p (I) or with 100 UM Gpp(NH)p (U). Total and blank incubations were carried out in duplicate in the absence or presence of 1 UM atropine respectively, with 50 ~1 of identical membrane suspension in a total volume of 1 ml. [3H]-L-QNB concentration was varieo between 80 pM and 2 nM. These experiments were replicated 5 times with similar results.

restored

(Figure

bound

RESEARCH COMMUNICATIONS

for

5-6

experiments

RT values

are

RT determined

in

determined expressed NaPi

buffer.

as

the

Vol. 113, No. 1, 1983

Figure

in

increases

(2

the

NaPi

shown).

Studies

were

to

determine Tris,

increase

in

[3H]-L-QNB an

in

alone. stable

in

GTP

and

of binding

(Figure

but

not

nucleotide to

the

GDP

and

Gpp(NH)p

100

PBS

in

their

PM.

for

a dose

the

R,

dependent

1 mM choline,

results

this in

alter

were

obtained

an ECSO adenine

NaPi

binding

with

30

more nM

nucleotides

283

(Figure

restoration

buffer

(Figure

in

measured at

lo-fold of

GN-mediated

measured

restoration

about

The

on

NaPi.

binding

not

[3~]-~-~~~

nucleotides

of

and

isotherms

of

caused

Similar

RT

binding

presence

determined

do

is with

the

2). in

levels

adenine

Gpp(NH)p

specificity

produced

binding,

GN and

[3H]-L-QNB

buffer,

at

in

the

the

concentrations

buffer.

RT

restore

analog

restoration restore

RESEARCH COMMUNICATIONS

reduces of

saturating of

NaPi

in

Tris

NaPi

GTP

and

choline analysis

influences

buffer,

GDP completely

measured

with

binding

the in

Similarly, Scatchard

out

increase

We examined

and

PBS

Tris,

RT measured

in

the

in

reflecting

1).

carried

measured

Gpp(NH)p

(Table

KD measured

not

nM)

AND BIOPHYSICAL

2 The influence of Gpp(NH)p on [3H]-L-QNB binding to atria1 membranes in the absence or presence of choline. Dose response curves for the effect of Gpp(NH)p on [3H]-L-QNB binding to atria1 membranes were constructed using 50 ~1 tissue homogenate in NaPi buffer in the absence (0) or the presence (a) of 1 &I choline. [3H]-L-QNB was 2 nM throughout and total and nonspecific binding were determined for each condition by the absence or presence of 1 UM atrogine. No effect on blank values was observed. Results are presented as the I' restoration from binding observed in the presence of choline without Gpp(NH)p additioti, to the binding observed in NaPi without choline addition. Typically, specific binding in the presence of choline was 1300 cpm/50 ~1 tissue (taken as O'/' restoration) and was restored to 2000-2200 cpm/50 ~1 tissue (approximately 80'1' restoration). Binding in NaPi buffer alone was 2200-2400 cpm and is defined as lOO'/' restoration. The values are the mean of 4 independent determinations. -+ S.E.M.

KD measured

(data

BIOCHEMICAL

Tris

to

in

NaPi

approximately potent

ATP,

buffer

GTP

GMP ADP

and

GTP

that

300

than 3).

3).

of

nM. in

fails AMP

The

its to have

no

Vol. 113, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

-log [Nucleotide] Figure

RESEARCH COMMUNICATIONS

(MI

3The influence of guanine nucleotides on [3H]-L-(INB binding in NaPi buffer or Tris. Dose response curves for Gpp(NH)p (o), GTP (I ), GDP (D), and GMP (A) were constructed using 2 nM [3H]-L-QNB with 50 ~1 membrane suspension incubated in a volume of 1 ml Tris Buffer. Dose-response curves constructed in NaPi buffer for Gpp(NH)p (0) and GTP (A) showed no effect on total or blank binding levels. Results are presented as the percent restoration from binding in Tris to the binding observed in NaPi buffer alone, as described in Figure 2. Values presented are the mean + SEM of 4 independent determinations.

effect

on [3H]-L-QNB

1O-8 M to lo4

binding

M (data

in Tris

not

or NaPi buffer

at concentrations

of

shown). Discussion

The major binding

finding

of the

to the mAChR is

present

absolutely

In PBS or NaPi buffer,

Tris.

C3H]-L-QNB

to atria1 the

the

isotherms

binding

choline

RT

or Tris

cannot

to

simply

apparent

GN regulation

on the

in the in

that

that

presence

to influence

whereas

observed

confirms

is on the

be explained

GN failed

levels

is

dependent

membranes,

GN restored

study

the

PBS

RT, and the

as a reduction

presence

alone.

action

the

of antagonist of choline

binding

in affinity

or Tris,

analysis

of GN in the action

of

of choline

Scatchard

or

presence

of

of

of the

nucleotides

of the

receptor

for

the

ligand. We previously Tris

which

distinct

regulates from

Approximately and Tris observations,

reported

presence

antagonist

previously one half

regulation

the

of an allosteric

binding

described of the

to the mAChR (8).

agonist

antagonist

or antagonist sites

in a non-competitive

we postulated

site

manner

a heterogeneity

mAChR.

284

appeared (8).

of antagonist

This binding sensitive

for

choline site

and

is

sites. to choline

Based on these sites

within

the

Vol. 113, No. 1, 1983 In this

report

an obligatory binding

we show that

feature

for

to the mAChR.

substitute

for

illustrated rats

BIOCHEMICAL

the expression

or Tris

for

canine

and mice and in atria

used by Burgisser effect

occupation

of this

(7)).

this

membranes,

and ventricles

et al.

site

and di-valent

in allowing

atria1

RESEARCH COMMUNICATIONS by choline

of GN regulation

None of the mono-

choline

here

AND BIOPHYSICAL

ions

also

of frogs

The widespread

present This

observed

in PBS can phenomenon,

in atria

(Rana pipiens;

biologic

is

of antagonist

expression. is

or Tris

the

expression

from species

of this

is evident. The obligatory

involvement

regulation

of antagonist

and models

(7,

We cannot allosteric

site

resides

yet

undefined

clarify

the

(9).

role

a re-examination

a further

constraint

data

site

nor the

exact

information

is

More

the mAChR, with

component

choline/Tris

present

regulatory

protein within

the

allosteric

suggests

and imposes

from

choline

GN regulatory

binding

8, lo),

determine

of the

receptor

allosteric

site

in GN

of available on plausible location

needed

of the

to determine protein,

complex, in cholinergic

data models.

mode of interaction

the GN regulatory

of the functional

of GN and this

the molecular

site

with if

the

this

or another as well

as

as to

transmission.

Acknowledgements We acknowledge gratefully the skillful technical assistance of Mrs. Colenda and Mrs. J. Simkins. This work was supported by grant HL-22675 A.S. and Medical Scientist Training Program grant GM-07309 (K.M.M.M.).

K. to

References 1.

Rosenberger, L.B., Yamamura, H.I., and Roeske, W.R. (1980) J. Biol. Chem. 255, 820-823. 2. Berrie, C.P., Birdsall, N.J.M., Burgen, A.S.U., and Hulme, E.C. (1979) Biochem. Biophys. Res. Comnun. 87, 1000-1005. Wei, J.W. and Sulakle, P.V. (1979) Eur. J. Pharmacol. 58, 91-92. i: Burgen, A.S.V. and Hulme, E.C. (1978) Mol. Pharmacol. 14, Birdsall, N.J.M., 723-736. 5. Birdsall, N.J.M., Hulme, E.C. and Burgen, A.S.V. (1980) Proc. R. Sot. Lond. 8207, 1-12. 6. Hulme, E.C., Berrie, C.P., Birdsall, N.J.M. and Burgen, A.S.V. (1981) Eur. J. Pharmacol. 73, 137-142. 7. Burgisser, E., DeLean, A. and Lefkowitz, R.J. (1982) Proc. Nat'l. Acad. Sci. 79, 1732-1736. 8. Sastre, A., Murphy, K.M.M. and Rusher, M.M. (1982) Biochem. Biophys. Res. Commun. 104, 383-388. 9. Rodbell, M. (1980) Nature 284, 17-22. 10. Hosey, M.M. (1982) Biochem. Biophys. Res. Commun 107, 314-321. 285