[3H] verapamil binding sites in skeletal muscle tranverse tubule membranes

BIOCHEMICAL

vol. 118, No. 1, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 239-245

January 13, 1984

[3H]

VERAPAMIL BINDING SITES IN SKELETAL MUSCLE TRANSVERSE TUBULE MEMBRANES

Jean-Pierre

GALIZZI,

Centre

Received

November

30,

Michel

FOSSET and Michel

LAZDUNSKI

Centre de Biochimie National de la Recherche Scientifique Facultg des Sciences, Part Valrose 06034 NICE CEDEX, FRANCE

1983

binding to muscle tubule membrane has the following SUMMARY : [3H]verapamil capacity Bmax = 50 * 5 pmol/mg properties. KD = 27 f 5 nM and maximum binding 05 protein. A 1 = 1 st ichianetry of binding was found for the ratio of binding sites. The dissociation cons[ Hlverapamil versus P H] nitrendipine tant found at equilibrium is near that determined fran the ratio of the rate constants for association (kl) and dissociation (kBl). Antiarrhytpic drugs like D600, diltiazem and bepridil are competitive inhibitors of [ Hlverapamil binding with KD values between 40 and 209 nM. Dihydropyridine analogs are apparent non canpetitive inhibitors of [ H]vera.pamil binding with half-maximum inhibition values (Koe5) between 1 and 5 nM. INTRODUCTION

: The most

pharmacological the

of

and clinical

dihydropyridine

like

D600,

[ 3Hlnitrendipine

and smooth

in frog

membranes

indeed

been found

muscle

membranes

[3H]nimodipine bit

skeletal

sites

than

other

and muscle

have

channel

with

at least

known tissues shown that

properties

(4-5).

verapamil

differing

(reviewed

muscle

of this

result

Pure

T-tubule

more

a blocker

from those

in nerve,

in the

calcium

transverse

membranes sites

than

have surface with

membranes

from

experiments

of voltage-dependent

of nitrendipfne

All

Copyright 0 1984 rights of reproduction

rab-

binding

and analogs 0006-291X/84

239

tubu-

was obtained

[3H]nitrendipine

Electrophysiological is

Receptors

voltage-dependent

binding

times

and compounds

in 2).

T-tubule

C3H]nitrendipine

50-100

include

been indentified

localized

(6-8).

properties

in 1 and 2).

shown that

skeletal

5) and a confirmation

have

these

or nimodipine

recently

are primarily

in

have an important

(reviewed

have

and d-cis-[3H]diltiazem muscle

with

membranes

Purified

to be richer (4,

Molecules

have

muscle

muscle

(T-tubules).

inhibitors

and bepridil

experiments

skeletal

channel

such as nitrendipine

[ 3 Hlnimodipine

Electrophysiological

lar

interest.

diltiazem or

skeletal

channels

calcium

derivatives

verapamil,

cardiac,

potent

on heart Ca 2+ (9,

10).

$1.50

by Academic Press, Inc. in uny form reserved.

Vol. 118, No. 1, 1984

BIOCHEMICAL

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1n the present work [3H]verapamil has been used to characterize and pharmacological properties rabbit

of its specific

the biochemical

binding site on purified

skeletal muscle T-tubule membranes.

MATERIALSANDMETHODS: T-tubule membraneswere isolated fran rabbit skeletal muscle as already described (11) and stored as in (5). Protein concentration was de ermined by the method of Hartree using bovine serun albumin as a standard.[ 3Hlnitrendipine binding experiments were carried out as previously descr&bed (5). Binding experiments with PHperapamil were done as described for [ Hlnitrendipine (5). In the routine assay, samples of membraneswere incubated at 10°C in 1 ml of a soluti n containing 50 mMTris-Cl buffer, pH 7.5 and the required concentration of P Hperapamil. The final concentratign of membraneswas 0.06-0.07 mg of protein/ml. In all binding experiments,[ Hlverapamil was added last. In standard equilibrium binding experiments, incubation lasted 70-80 min. Incubations were stopped by rapid filtration of 400 l.ll samples of the incubation mixture through polyethylene imine treated whatman GP/C glass fiber filters, under reduced pressure. The filters were immediately washed twice with 8 ml of a cold solution containing 200 mMcholine chloride and 20 mMTris-Cl at pH 7.5. Experiments in duplicate were systematically carried out. For measurements of nonspecific binding, unlabelled veryamil was present in the medium at a final concentration of 10 PM. Binding of [ Hlverapamil to the filters alone was shown to be non-negligible and was displacable in the presence of an excess of verapamil (10uM). Therefore, for each measurement in without membranes a given assay, a control was carried out with the filters under the sameconditions as in experiments with T-tubule membranesand this control was substracted from total binding. The filters used in the experiments were psetreated during 10 min with 0.05 % polyethylene3imine in order to minimise [ Hlverapamil binding to filters. Inhibition of [ Hlverapamil binding by different drugs was measured un$ar equilibrium conditioy as described above, in the presence of 039-1.0 nM [ Hlverapamil. N-methyl-[ Hlverapamil at 75 Ci/ mmol and S-methyl-[ Hlnitrendipine at 85 Ci/mmol were obtained from NEN. All other canpounds used were obtained as described in (5).

RESULTS ANDDISCUSSION: [3H]verapamil binding to T-tubule membranes. Fig. 1 A illustrates

a typical

equilibrium

T-tubules membranes.The linearity shows specific of protein

of the Scatchard plot shown in Fig. 1A (inset)

binding to a single class of sites and gives values of 53 pmol/mg

for the maximumnuaber of [3H]verapamil

the equilibrium Specific

binding experiment of [3H]verapamil to rabbit

dissociation

sites (Bm,,) and 27 nM for

constant (KD) of the verapamil-receptor

binding of r3H]verapamil varied

linearly

canplex.

with increasing concentrations

of pure T-tubule membranesin the range of 30 - 90 ug of membraneproteins/ml (not shown), Measurments of [3H]nitrendipine lel and we confirmed our previous results

binding were carried out in paral-

(6, 7) giving a Bmax = 55 i: 6 pmol/mg

protein and a KD value of 1.8 + 0.3 t-&i. Thus a 1:l stoichiometry C3H]verapamil and L3H]nitrendipine

exists between

binding sites on T-tubule membranes. 240

BlOCHEMlCAL

Vol. 118, No. 1, 1984

time

incubation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(min)

incubaticn

the(min)

Fig.

1. Equilibrium binding of [3H]verapamil to T-tubule membranes and kinetics of association and dissociation. Equilibrium binding was measured using[3H]verapamil prepared at 11 Ci/mmol and 0.07 mg/ml of membranes (pH 7.5 and 1O’C). A, Binding to T-tubule membranes in the absence (a,~ ) or presence (b.0 ) of 70 UM unlabelled verapamil and canparative binding to GF/C filtera, alone, without membranes, in the absence (c, A) OK presence (d, l ) of 10 UM unlabelled verapamil. The specific binding canponent3ia calculated as (a-b) - (c-d). A, inset, Scatchard plot for the specific [ Hlverapamil binding component. Noxat GF/B filters gave 5 times more binding to filters than GF/C filters and are to be avoided in experynts of that kind. B (o)Mrect association [ Hlverapamil binding’: Concen$.inetics corresponding to specific tration of I Hlverapamil : 0.8 &I (75 Ci/mmol) and concentratLon of T-tubule membrane : 0.070 mg of protein/ml, pH 7.5, 10°C. Non-specific binding was measured as usual in the presence of 10 uM verapamil, and subetracted fraa total binding. (@) pseydo-first order representation of the data. X, extent (in X) of the specific [ Hlverapamil association to T-tubule membranes. (I, After equilibrium has been reached (60-70 min) and checked to be stable for more than 2 h, the dissociation of the complex vas triggered by a ZO-fold dilution in a 50 I&! Tris-Cl buffer at pH 7.5. (0) time coureo of [‘Blverapamil signiflcally bound to its receptor. (0) first-order representation of the data.

Association r3H]verapamil were tor

carried concentration

and to out

dissociation

T-tubule with

kinetics.

membranes

are

a [3H]verapamil [ E,$

of

3.7

r&f.

Typical shown

concentration The

maximal

241

in

association Fig.

1B.

[$I concentration

These of

kinetics

of

kinetic

studies

0.8

nM and

of

specifically

a recep-

Vol. 118, No. 1, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

bound

13H]verapamil

0.082

nM. Consequently,

to pseudo [LR]~S

the

first-order

(12)

the concentration

constant kl

[LRJR which

dilution

order

s-l.

kinetics.

Dissociation

isKD

=

results

pamil,

of the

for

molecules

nes.

Half-maximum

Ko 5 = 25 nM. Under . of 0.9 - 1.1 nM which

with

KD for

KD values

(KD = 50 nM).

canplex)

unlabelled

lower

found

from

T-tubule

and Table

I show that

for

association

binding

the

membranes

with

of

reaches

90 X. The molecule

described

agonist

(opener)

ding

with

an apparent

inhibition

channel constant

242

on Ca

2+ channels

observed

diffe-

membraat

concentration constant

equilibrium is

vera-

of these

dissociation

value

I show

including

to T-tubule

KD of

dissociation

in good agreement

by bepridil,

family

apparent

[3H~erapamil

(13)

studies.

2A and Table drugs

is

data

binding

also

(KoS5)

as the

data

D600 and

40 and 200 nM (Table

1 and 5 TIM. Maximum inhibition

calcium

Fig.

formed

between

BAY K 8644,

from kinetic

active

of the dihydropyridine membranes

of 6.9 min

(Kg = 27 nM) or from kinetic

are between

to T-tubule

of the

was

[3averaPamil

the

This

binding

molecules

the

of

of dissociation

binding

KO.5 rep resents

equilibrium

The KD values

with

than

a value

followed

concentrations

(a

rate

dissociation

equilibrium

verapamil

complex.

1B gives

order

by a 20-fold

drugs

conditions

verapamil-receptor

Diltiasem

[3H]verapamil

[3H]verapamil

second

was obtained

membranes.

inhibited

30 times

the

of antiarrhythmic

experimental

the verapamil-receptor constant

group

for

the

and other

Increasing

is

where

KD calculated

and bepridil.

the

x t x[$][R~]/[Llt]~

of dissociation

with

to T-tubule

inhibition

according

in Fig.

constant

constant

verapamil

a first

gradually

plotted

1C shows that rate

1B was only

were

t and kl, plot

agreement

binding

diltiazem

rent

kl

to a half-life

equilibrium

of unlabelled

obtained

D600,

Fig.

corresponding

on r3H] verapamil the

association

at time

of the

50 nM, in approximate

The effect

for

of [3H]verapamil

canplex.

The value

The value

data

The semi-logarithmic

k -1 = 1.6 x 1O-3 s-l at 1O“C.

to 100 X in Fig.

: Ln C~lE/[LRIE-[~]=

of the preformed

first

kinetic

of the canplex

of association.

= 3.3 x lo4 M-l

correspond

also

I).

Fig.

2B

inhibit

K. . 5 concentrations binding first

inhibits of 30 nM.

by these

drugs

dihydropyridine [3H]verapamil

bin-

BIOCHEMICAL

Vol. 118, No. 1, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2+ binding to 2. Effects of different Ca -channel blockers on ] 3H]verapamil T-tubule membranes. A, Inhibition of ['H]verapamil (0.9 nM at 75 Ci/ D600, diltiazem and bepridil. Binding mm013 binding by unlabelled verapamil, of [ H]verapamil to T-tubule membranes (0.06 mg/ml) was measured under equiconcenlibrium conditiona at pH 7.5 and IO'C, in the presence of the indicated tratlons of veryamil (0). bepridil (A), D600 (A) and diltiazem (0). E, Inhibition of [ HI verapamil ( 0.9 nM) binding by nitrendipine, nimodipine, nifedipine, nisoldipine and BAY K 8644 as described in A. Binding was measured in the presence of indicated concentratioea of nitrendipine (A), nimodipine (m ), nifedipine (0). nisoldipine (0) and BAY K 8644 (A). Fig.

Fig. binding

3A shows a family to T-tubule

nitrendipine. with dipine

of equilibrium

concentrations

as an apparent membranes. membranes

membranes

One observes

no change

Fig.

of Scatchard

are

noncanpetitlve 3B shows

in the presence

in the

plots

presence

a decrease

increased

constant

fran

inhibitor plots

of increasing

zero for

concentrations

binding (s

capacity

C3H]verapamil

concentrations

of (Bmax)

= 27 nM) when nitren-

to 1.7 nM. Nitrendipine

of [3H]verapamil

243

3H]verapamil

of increasing

of the maximal

dissociation

Scatchard

describing[

behaves

binding

to T-tubule

binding

to T-tubule

of diltfazem.

One ob-

Vol. 118, No. 1, 1984

8lOCHEMlCAl

AND 8lOPHYSlCAL

RESEARCH COMMUNICATIONS

TABLE I. Inhibitors of [3h] verapamil binding to T-tubule membranes Inhibition constant KD or K0.5 Wf) Verapamil Bepridil MOO Diltlazem Nitrendiplne Nimodipine Nifediplne Nisoldiplne Agonist BAY K 8644

serves an increase of the equilibrium binding capacity increased

fra

25 40 70 200 1.4 1.6 3.5 5 30

dissociation

(Bmax = 50 pmolfmg protein)

when diltiazem

(KD = 29 nM),

to 210 nM (apparent

behaves as a ccrmpetitive inhibitor

for [3H]verapamil

membranes. Fig.

zero

constant with no change of

in the presence

no change

of binding is

Bepridil

increased

also

KD = 61 nM).

are

Diltiazem

binding to T-tubule

3C shows Scatchard plots of [3H]verapamil binding to T-tubule

membranes

tration

concentrations

behaves

of increasing

capacity fran

zero

(B,,,

concentrations

of bepridil.

= 52 pmol/mg protein)

when bepridil

(KD = 28 nM) to 52 nM (apparent

as a canpetitive

inhibitor

Fig. 3 . Eouilibrium

for

There

[3H]verapamil

is

concen-

KD = 55 nM). binding

bindins of [3H]verapamil to T-tubule membranes in the absence and in the presence of nitrendipine. diltiazem or bepridil. out at pH 7.4 and 1O'C with 0.06 mg of membranes/ Binding assays were carried ml.. &, Scatchard plots in the absence (0) and in the presence of 1 nM (A) 1, Scatchard plots in the absence (0) and in the or 1.7 nM (m) nitrendipine. presence of 100 nM (A) or 210 nM (u) diltiazem. c, Scatchard plots in the absence (0) and in the presence of 24 194 (A) or 52 nM (D) bepridil. 244

BIOCHEMICAL AND BlOPHYSfCAL RESEARCH COMMUNlCATlONS

Vol. 118, No. 1, 1984 to T-tubule

membranes.

The observation

competitive

inhibitors

of [3H]verapamil

that

verapamil,

diltiazem

blockers

because

binding

produced

by diphenylalkylanine

active

molecules

like

have been

membranes.

veratridine

types

like

himbine,

nists

prazosin

like

remark dil

atropine

experiments

Ca2+ channels

the

in

work

for

of these

binding

of the sodium

effecters

(1 pM)

like

phencyclidine

Finally verapamil

it

is

(4 FM),

tran-

propranolol,

yo-

and thienyl-

of interest

antagoto

(Kd = 25 nM and beprl-

of 30 nM found molecules

channel

of different

(1 I.IM) and cholinergic

(1 PM).

to EC50 values effects

pharmacologically

(10 PM) or apamine

phenobarbital

of calcium

of E3H]nitrendipine

effecters

blockers

as

the proposal

on [3H]verapamil

(1 PM) like

this

with

Many other

(10 PM),

adrenergic

like

found

for

(14).

behave

same family

inhibition

classical

and d-tubocurarine

affinities

is consistant

to have no effect

and psychotomimetics

(Kd = 40 nM) are similar

logical

the

tetraethylammonium

(10 PM),

and bepridil

to the

drugs

found

antiepileptic

that

reverse

They include

like

diazepam

phencyclidine,

all

belong

(100 $iM) and tetrodotoxin

of K+ channels

quizer

they

diltiazem

binding

and bepridil

channel

to T-tubule

that

from electrophysio-

on frog

skeletal

(10).

ACKNOWLEDGEMENTS : We are grateful to M. Tomkowiak for expert technical assistance and to Dr. Traber, Dr. Horstmann, Dr. Zellerhoff, Dr. Mijller and Dr. Schramm fran Bayer A.G., FRG, for providing us dihydropyridine derivatives. This work was supported by the Centre National de la Recherche Scientifique. REFERENCES : 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. -12. 13. 14.

Fleckenstein, A. (1977) Ann.Rev.Pharmacol.Toxicol. x, 149-166. Janis, R.A., and Triggle, D.J. (1983) J.Med.Chem. 26, 775-785. Almers, J.A., Fink, R., and Palade, P.T. (1981) J.Physiol. 312, 177-207. Fosset, M., Jaimovich, E., Delpont, E., and Lazdunski, M. (1982) Eur.J. Phannacol. 86, 141-142. Fosset, M., Jaimovich, E., Delpont, E., and Lazdunski, M. (1982) J.Biol. Chem. 258, 6086-6092. Ferry, D., R., and Glossmann, H. (1982) FEES Lett. 148, 331-337. C.B. (1983) Naunyn-Schmied.Arch. Glossmann, H., Ferry, D.R., and Boschek, Phannacol. 323. l-11. Glossmann, H., Linn, T., Rombusch, M., and Ferry, D.R. (1983) FEBS Lett. 160, 226-232. Lee, K.S., and Tsien, R.W. (1983) Nature=, 790-794. Kerr, L.M., and Sperelakis, N. (1982) J.Pharmacol.Exp.Ther. 222, 80-86. Rosemblatt, M., Hidalgo, C., Vergara, C., and Ikemoto, N. (1983) J.Biol. Chem. 256, 814C-8148. P.B. (1981) Life Sci. 2, 313-330. Weiland, G.A., and Molinoff, Schramm, M., Thomas, G., Towart, R., and Franckoniak, G. (1983) Nature 303, 535-537. Murphy, K.M.M., Gould, R.J., Largent, B.L., and Snyder, S.H. (1983) Proc. Natl.Acad.Sci.USA 0, 86s864 245