Carboxyl residue(s) at the ligand-binding site of rat muscarinic receptors

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ] 2 0 3 - ] 2 0 8

November 15, 1988

CARBOXYL RESIDUE(S) AT THE LIGAND-BINDING SITE OF RAT MUSCARINIC RECEPTORS Ronit Galron and Mordechai Sokolovsky Laboratory of Neurobiochemistry Department of Biochemistry The George S. Wise Faculty of Life Sciences Tel Aviv University, Tel Aviv 69978, Israel Received August i, 1988

Chemical modification of muscarinic receptors of rat cerebral cortex, brain stem and atria by a carboxyl-group-specific reagent, namely trimethyloxonium ion (TMO +) reduces the number of tritium-labeled antagonist- and agonist-binding sites in a dose-dependent way. No such effect is observed when modification is carried out in the presence of atropine, oxotremorine or carbamylcholine. These findings suggest that TMO + specifically methylates the carboxyl residue(s) positioned at the binding site in members of the MI and M2 receptor family. ~ 1988AcademicPress, Inc.

Most

mechanistic formulations of the binding of cholinergic ligands to

muscarinic receptors assume the presence of essential carboxyl groups at the site

which

interacts with the typical "cationic head", i.e. the quaternary

nitrogen of the ligand. In previous attempts to modify proteins

using

tri-

alkyloxonium salts (I-3) only a limited number of carboxyl groups were found to react, indicating a certain degree of specificity

on

the

part

of

the

reagent. Rawn and Lienhard (4) and Chao et al. (5) used the trimethyloxonium ion-((CH3)30+), (TMO+), to modify acetylcholinesterase and the nicotinic receptor,

reasoning that because of the similarity in size and charge between

TMO + and the quaternary ammonium ion the reagent should be directed "cationic

head"

binding

site.

Preferential

to

the

chemical modification - i.e.

esterification of carboxyl residues - was indeed observed in these proteins. Freeman

et

al.

(6) reported that methylation by TMO + of critical carboxyl

groups in the left atrium of the guinea pig leads to loss In

this

paper

we

report

of

excitability.

on modification of anionic binding sites in the

muscarinic receptor of rat brain and atrium by

the

pseudoaffinity-labeling

reagent TMO +. The brain regions selected for study were the cortex, which is representative of the MI subtype, and the brain stem which, like the atrium, represents the M2-1ike subtype (7, 8). MATERIALS AND METHODS Trimethyloxonium-tetrafluoroborate was purchased from Fluke AG (Buchs, Switzerland) and stored at -20°C under nitrogen. [3H]4NMPB (45 Ci/=~ol) was

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prepared by catalytic exchange as described (9). [3H]AcCh (86 Ci/mmol), 97% purity was purchased from New England Nuclear (Boston, MA). Carbamylcholine chloride, oxotremorine, acetylcholine, and atropine sulfate were from Sigma (St. Louis, MO). Tissue Preparation. Adult male rats of the CD strain were obtained from Levinstein's Farm (Yokneam, Israel) and maintained in an airconditioned room at 24±2°C for 14 h (0500-1900) under fluorescent illumination and in darkness for i0 h. Food from Assia Maabarot Ltd. (Tel Aviv, Israel) and water were supplied ad libitum. Rats aged 3-4 months and weighing 190-250 g were decapitated (between 0900 and i000 h), and the required brain regions were immediately dissected out in a cold room. Tissues (pooled from 2-3 rats for cortex and from 5-6 rats for brain stem) were homogenized in 0.32 M sucrose using a glass Teflon homogenizer. The homogenates were centrifuged at i000 g for I0 min, and the supernatant was further centrifuged at 15000 g for 30 min and the resulting membrane pellet (P2 fraction) was used for the chemical modification and binding experiments. Atrial preparations (pooled from 5-6 rats) were prepared similarly except that they were homogenized at setting 5 on an ultra-Torrax (Ika-Werk Instruments, West Germany) with three 15-s bursts separated by 30-s pauses. Modification of the Muscarinic Receptors by TMO +. Aliquots of TMO + dissolved in acetonitrile (25 mg/100 ~L) were added to 5 nil of membrane fragments (P2 fraction) in Krebs-Henseleit buffer containing 25 mM Trischloride, pH 7.4. The reaction was carried out and maintained at 0°C in an ice bath for 30 min with gentle shaking, and then stopped by washing of the membranes twice with ice-cold Krebs-Henseleit buffer. The final pellets were resuspended in the same buffer. Because of the instability of TMO + in aqueous medium we refer throughout the manuscript to the measured weight of the reagent rather than to its molar concentration. !SH]AcCh Bindin~ AssayS(10). Aliquots (20 ~L) of freshly prepared membranes were added to tubes containing 20 ~L of modified Krebs buffer, 200 ~M DFP, and various concentrations of [3H]AcCh. After incubation for 1 h with gentle shaking at 25°C, 3 mL of ice-cold modified Krebs buffer was added and the contents of the tubes were filtered through GFIC filters. The filters were irmnediately washed with an additional 3 mL of buffer. Filters were counted for tritium by using a scintillation cocktail (Hydroluma) and a scintillation spectrometer (LKB-1218) at 48% efficiency. An incubation period of 1 h was chosen, since after this period even the lowest concentration of [3H]AcCh employed has reached equilibrium. Ice-cold buffer was used for washes to prevent dissociation of [3H]AcCh from the receptors, since at 0°C the dissociation half-time is relatively slow (90 s) (i0). !3H]4NMPB bindin~ (9). Aliquots (50 ~L) of the preparation were incubated for 30 min at 25°C with various concentrations of [3H]4NMPB (0.1-10 nM) in 1 mL of buffer. Binding was terminated by filtration through GF/C filters and washing 3 times with 3 mL of ice-cold buffer. Nonspecific binding was determined with 10 VM atropine. !3H]4NMPB Displacement. Aliquots (50 ~L) of the preparation were added to tubes containing 1 ml of Krebs-Henseleit buffer containing 2 nM [3H]4NMPB and 50 ~L of agonist (10-9-10 -2 M). After incubation for 30 min with gentle shaking at 25°C, binding was terminated by filtration. RESULTS AND DISCUSSION The effects of various concentrations of TMO + on the binding properties of the antagonist [~H]4NMPB to muscarinic receptors in the rat cortex, brain stem and atrial preparations were examined. Figure strates

the

binding to muscarinic receptors of cortical is a

decrease

I

(left

panel)

demon-

concentration dependence of the TMO+-induced loss of [3H]4NMPB

in

binding

homogenates.

The

main

effect

site density, with no effect on the K D value of

the remaining binding sites. For example, 50%

1204

reduction

in

binding

(Is0)

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cortex.

Atria.

Brainstem.

100 I80 0 Z Z

60

m Z qr

2O

.,I,-

l

I

I

I

I

0.2

0.4

0.6

0.8

~ 1.0

I

I

0.2

0.4

I , 0.6

I

I

I

I

I

I

I

0.8

1.0

0.2

0.4

0.6

0.8

1.0

{ mg/m, )

T RIMETHYLOXONIUM - TETRAFLUOROBORATE

Figure 1 Effect of TMO+ treatment on muscarinic binding to rat cortical, atrial and brain stem preparations. Membranes (25 mg of protein/mL) were treated for 30 min at 0°C in the presence (o, A) and absence (o) of atropine (i0 ~M) (o) or oxotremorine (i m_M) (&) with the indicated concentrations of TMO + in Krebs buffer, pH 7.4. Results in this figure represent a typical experiment. Each experiment was carried out at least four times.

occurred

at

0.02

mg/mL

of

TMO +, and the K D values before and after such

modification were 0.7 and 0.9 nM respectively. The time the

reaction

is

course

shows

that

rapid, with most of the effect already induced within the

first i0 min. Accordingly, in all furter experiments a period of 30 min

was

allowed for reaction of TMO + with the muscarinic receptors. Similar

results

were

obtained

upon modification of atrial and brain

stem preparations with TMO + (Fig. i). The cortical preparation ever

to

seamed

how-

be more sensitive to reagent concentration: thus, 50% reduction in

binding occurred at about 0.02 mg/mL of TMO + for this preparation, while the corresponding

concentrations

for

brain

stem and atrial preparations were

about 0.i mg/mL. Again the K D values for the remaining

binding

sites

were

unchanged, i.e., i.i nM and 0.85 nM for brain stem and atrium, respectively, in the control receptor preparations as compared to 1.2 and i.i

nM

respec-

tively in the modified receptors preparations. It

is

important

to

note that when 10 ~M atropine, 1 mM oxotremorine

or 10 mM carbamylcholine was added to the reaction mixture 10 min the

addition

of

TMO +,

prior

to

the loss of binding sites due to TMO + modification

was completely prevented even at the highest TMO + concentration. This, finding

supports

the contention that TMO + modifies a residue(s) at the binding

sites both for antagonist and for agonist of the M1 and

the

M2

muscarinic

receptors. We

cannot

yet

directly demonstrate the product formed as a result of

TMO + modification of musearinic receptors. From this

the

known

specificity

of

reagent, the most likely candidate would appear to be a carboxyl resi-

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

due. In principle it should be possible to demonstrate the

formation

of

a

methyl ester by the use of radiolabeled TMO +. Unfortunately, attempts in our laboratory as well as in two commercial laboratoriess have met

with

little

success in the preparation of [SH]TMO+ with high specific activity. Freeman et al. (6) showed that the methylation of TMO +

carboxyl

groups

by

in the guinea pig atrium abolishes excitability and contractility, and

that [SH]QNB-binding sites are lost during methylation. These in agreement

with

the

results

are

reported here. However, their finding that

atropine and QNB offer little protection against the following

findings

loss

of

excitability

methylation, and that acetylcholine cannot protect the muscarinic

receptor from methylation, led them to suggest that the muscarinic receptors are

not

involved

in

the

maintenance of excitability. The discrepancy in

the results of protection experiments carried out by these authors and selves

might

be

attributed

our-

to the experimental conditions employed: they

used intact organs whereas we used homogenates, and they

used

concentrations

concentrations

of

TMO +

(25

mM)

and

relatively

low

much

higher of

protectors (I0 ~M). The effect of TMO + on agonist binding can be evaluated binding

of

[SH]AcCh

by

(a)

direct

to the high-affinity sites (i0), and (b) displacement

experiments aimed at determining the characteristics of binding of

agonists

such as AcCh and carbamylcholine to the remaining sites, i.e., those unmodified by TMO +. As shown in Table I, TMO + induced the loss of [SH]AcCh binding to

muscarinic

receptors

of

atrial

and brain stem preparation in a dose-

Table 1 3

Specific binding of [SH]4NMPB and [ H]AcCh, and Is0 values of unlabeled agonist to muscarinic receptors in rat atria and brain stem before and after modification with trimethyloxonium-tetrafluoroborate Specific binding of [SH]4NMPB

Specific binding of [SH]AcCh 150 (l~l) from disBmax KH placement experiment

TMO+ (mg/mL)

fmol/mg

Atria: control 0.05 O.1 0.25

360±86 270±56 165±30 94±16

i00 75±15 46±12 26± 5

175±43 125±22 89±15 56±14

Brain stem: control 430±87 0.05 310±71 0.1 220±45 0.25 123±29

I00 73±15 50±12 30±81

189±42 160±23 108±17 81±15

%

fmol/mg

%

(nM)

Carbamylcholine

i00 70±15 50±10 32± 7

20±8 23±7 20±8 25±6

0.52±0.06 1.2 ±0.Ii 1.5 ±0.15 1.3 ±0.12

0.5±0.08 3.0±0.30 2.8±0.32 3.1±0.30

i00 86±15 55±10 44± 9

24±7 26±6 24±8 27±6

2.0 8.0 6.0 8.0

2.8±0.32 5.0±1.25 6.6±1.30 8.0±1.50

±0.43 ±1.53 ±1.30 ±1.52

AcCh

Mean and SD values are shown for at least 3 experiments for each preparation. Assays were performed as detailed in Materials and Methods. Protein was determined by the Lowry method using bovine serum albumin as a standard.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

dependent manner which was very similar to that observed antagonist

sites,

for

the

loss

for agonist and antagonist binding. The displacement curves for the of

AcCh

binding

and carbamylcholine binding to modified cortical preparations were

very similar to those of the control. Similar behavior was seen with and

brain

stem

changes

atrial

preparations although, as shown in Table i, slight changes

were observed as exemplified in changes in the 150 these

of

indicating that the same carboxyl residues are essential

values.

The

fact

that

were small indicates that both the high and the low affinity

agonist sites not modified by TMO + possess

binding

properties

similar

to

those of the native receptors. Results

of

other

chemical

modifications

are

in agreement with the

results reported here for the modification of carboxyl residue(s). Hulme al.

(Ii)

reported

recently that the affinity-labeling reagent [3H]propyl-

benzylylcholine mustard (PrBCM) alkylates a shown

by

peptide

et

mapping

to

reside

carboxylate

group,

which

was

probably within the second or third

putative transmembrane region. Furthermore, dicyclohexylcarbodiimide b l o c k e d muscarinic

binding

sites

in

a

time-dependent and antagonist-protectable

manner (ii). Also, a group with a pKa of 6.2, which is thought to be characteristic

of

carboxylates

in

a

hydrophobic environment, is assumed to be

involved in the binding of muscarinic antagonists (ii). After comparing amino

the

acid sequences of MI and M2 receptor subtypes with those of rhodopsin

and ~-adrenergic receptors, these authors proposed that Asp 71 is the pri3 candidate for alkylation by [ H]PrBCM, with Asp 105 and Asp 99 as

mary

possible but less likely contenders. Of relevance to the above findings are directed

mutagenesis

experiments

aimed

the

results

of

required for ligand binding to the ~-adrenergic receptors therein):

(a)

Substitution

of

an

Asn

recent

site-

at the identification of residues

for

Asp-ll3

((12)

and

refs.

in the ~-adrenergic

receptor resulted in a decline in ligand binding. The most

likely

explana-

tion is an interaction between the protonated amino group of the ligand with the carboxylate of Asp-ll3. Moreover, replacement of Asp 79 had no effect on antagonist

binding

by

the

receptor,

but

the

affinity for agonists was

reduced by one order of magnitude. Asp 79 in the 8-adrenergic receptor responds

to

cor-

Asp 71 in the muscarinic receptor. (b) The ligand binding site

contains the transmembrane region of the ~-adrenergic receptors. (c) On

the

basis of the structural homology between the 8-adrenergic and the muscarinic receptors, Strader et al. (12) proposed a similar role for Asp muscarinic

receptor

to

121

i.e. involvement in binding of the quaternary amino group ligands. assignation

of

in

the

the role of Asp 113 in the 8-adrenergic receptors, However,

this role to Asp 121 in the muscarinic receptor would imply

that the ligand binding site is located

1207

deep

inside

the

membrane,

which

Vol. 156, No. 3, 1988

would

be

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

an unexpected result since the general belief is that the binding

site has to be at least partly exposed to the extracellular side, especially when

the

ligand is a charged and hydrated molecule such as AcCh. Interest-

ingly, if one considers the possibility of an error in this

role

to

Asp

the

assignation

of

121, and its assignation instead to Asp 105, this would

localize the ligand binding site at a more accessible position in

the

mem-

brane, while at the same time conforming to the proposed structural homology between the 8-adrenergic and the muscarinic receptors. It

should

be

noted

that the possibility of an hydrophilic pocket located deep inside the transmembrane region can not be discounted.

REFERENCES I. 2. 3. 4. 5. 6. 7. 8.

9.

10. 11.

12.

Parsons, S., Tao, L., Dahlquist, E.W., Borders, C.L., Groff, T., Racs, J., and Raftery, M.A. (1969) Biochemistry 8, 700-712. Nakayama, H., Taniyama, K., and Kanaoka, Y. (1970) Biochem. Biophys. Res. Commun. 40, 537-541. Paterson, A.K., and Knowles, J.R. (1972) Eur. J. Biochem. 31, 510-517. Rawn, J.D., and Lienhard, G.E. (1974) Biochem. Biophys. Res. Cor~un. 56, 654-660. Chao, Y., Vandlen, R.L., and Raftery, M.A. (1975) Biochem. Biophys. Res. Co=~un. 63, 300-307. Freeman, S.E., Dawson, R.M., Balden, M.P., and Gray, P.J. (1986) J. Mol. Cell Cardiol. 18, 99-108. Bonner, T.I., Buckley, N.J., Young, A.C., and brann, M.R. (1987) Science 237, 527-532. Kubo, T., Maeda, A., Sugimoto, K., Akiba, I., Mikami, A., Takahasi, H., Haka, T., Haga, K., Ichiyama, A., Kangawa, K., Matsuo, H., Hirose, T., and Numa, S. (1986) FEBS Lett. 209, 367-372. Kloog, Y., Egozi, Y., and Sokolovsky, M. (1979) Mol. Pharmacol. 15, 545-558. G u r w i t z , D., K l o o g , Y . , and S o k o l o v s k y , M. (1983) Mol. P h a r m a c o l . 288, 297-305. Hulme, E.C., Wheatley, M., Curtis, C., and Birdsall, N.J.M. (1987) In: Muscarinic Cholinergic Mechanism (Cohen, S. and Sokolovsky, M., eds.) pp. 192-211, Freund Publ. House, London. Strader, C.P., Sigal, I.S., Register, R.B., Candelore, M.R., Rands, E., and Dixon, R.A.F. (1987) Prec. Natl. Acad. Sci. USA 84, 4384-4388.

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