Vol. 182, No. 2, 1992 January 31, 1992

BIOCHEMICAL

BIOCHEMICAL

Received

RESEARCH COMMUNICATIONS Pages 947-952

CHARACTERIZATION OF BINDING SITES FOR DIHYDROPYRIDINE AND o-CONOTOXIN IN BRAIN OF ADULT CHICKEN

A. Zgaga-Griesz, University

AND BIOPHYSICAL

R. Woscholski,

P. Straub,

of Freiburg, Institute Gddecke AG, Mooswaldallee December

20,

of l-9,

H. Hug,

Molecular Cell 7800 Freiburg,

D. Marmel Biology, FRG

c/o

1991

Binding studies using the calcium channel blockers Summary: o-conotoxin and dihydropyridine revealed a rather equal amount of binding sites in brain from adult chicken. The o-conotoxin binding sites could be solubilized using digitonin, without substantial loss, whereas a great decrease in dihydropyridine binding sites was observed, indicating that both types of binding sites have different sensitivity to solubilization. In contrast to ion exchange chromatography where both binding sites comigrated, glycoprotein affinity chromatography led to a different partition of the binding sites in the flow through and eluate fractions. Our results indicate that both types of calcium channel blockers bind to different targets in adult chicken. 0 1992 Academic Press, Inc. Voltage dependent calcium channels (VDCC) play a key role in excitation-contraction and excitation-secretion coupling. With respect to their electrophysiological properties the VDCC have been classified as L-, N- and T-type channels (1). L-type specific calcium channel blockers like 1,4-dihydropyridines (DHP) e. g. PN 200-110 were useful1 tools for the characterization, purification and structure analysis of this channel type from different tissues (2, 3, 4). o-Conotoxin (GVIA), a poisonous peptide produced by the marine snake Conus geographus blocks the N-type and the L-type VDCC from neuronal tissue, as shown by electrophysiological techniques (5). Previous work using both VDCC-blockers revealed that brain tissue from juvenil chicken contains 5-times more GVIA- than DHP-binding sites (6). However, both types of binding sites from adult chicken are much less characterized. 1

To whom correspondence

should

be addressed.

Abbreviations: omega-conotoxin (GVIA), 1,4-dihydropyridines (DHP), Voltage dependent calcium channels (VDCC), wheat germ glutinine (WGA). 0006-291x/92 947

ag$1.50

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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BIOCHEMICAL

AND

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We therefore investigated the amount GVIA and DHP in brains from juvenil chicks types of binding sites using chromatographic

MATE-S

AND

RESEARCH

COMMUNICATIONS

of binding sites and characterized techniques.

for both

-0DS

The drugs and chemicals used and their sources are as folWGA-Sepharose 6MB (Pharlows: Digitonin (Sigma): DEAE-Sepharose, GVIA (Peptide Institute): ['H]PN 200macia); PN 200-110 (Sandoz), Adult chicken were received from a local 110, [""J]GVIA (Amersham). hatchery and immediately used for preparation of brain membranes as previously described (7). brain membranes DHP- and GVIA _'bind i na to u&xpbranes : Chicken were diluted to a final concentration of 4 mg/ml or 0.1 mg/ml for DHP or GVIA binding, respectively and incubated with the indicated concentrations of ['H]PN 200-110 and ["'"J]GVIA, according to the methods of Curtis and Catterall (8) or Barhanin et al. (9), respectively. VDCC-blockers bound to the membranes were collected using rapid filtration throught Whatman GF/C filters (postlabel Nonspecific binding was obtainded in the presence of 0.4 method). PM unlabeled PN 200-110 or 9 nM unlabeled GVIA, respectively. Specific binding was calculated as the difference of the determined total and nonspecific binding. Protein concentration was measured according to Bradford (10). Solubilizationofmembranes : Brain membranes were solubilized using 1.25 % (w/v) digitonin according to Curtis and Catterall (8). GVIA-binding was performed as described by Yamaguchi et al. (11) using gelfiltration for collection of the GVIA bound to solubilized membranes. DHP-binding of the solubilized membranes could not be performed using postlabel techniques as described for the membranes, because this type of binding sites is sensitive to solubilization (see Results and Discussion). We therefore labeled membranes with ['H]PN 200-110 prior to solubilization (prelabel method) as described above. In order to obtain the specific DHPbinding the membranes were divided in equal aliquots. One aliquot was labeled only with ['H]PN 200-110 (representing total binding), whereas the other aliqout was labeld with [3H]PN 200-110 in presence of 2 pM unlabeled PN 200-110 (representing nonspecific binding). Solubilized membranes were directly used for liquid scintillation counting for determination of the bound [3H]PN 200-110. Anion emue chromatogranhv: Membranes were labeled with [3H]PN 200-110 or [""J]GVIA (for nonspecific binding: with the corresponding unlabeled VDCC-blocker) prior to solubilization using the described prelabel method. The solubilized membranes were loaded onto 1 ml DEAE-Sepharose columns. Each column was washed and then stepwise eluted with increasing NaCl concentrations as indicated. DHP- and GVIA-binding was determined by counting aliquots with and without liquid scintillation, respectively. . * Afflnltv Membranes were divided in equal aliquots. One aliquot was used directly for solubilization, whereas the other aliqouts were used for DHP-labeling prior to solubilization. Each aliquot of solubilized membranes was loaded onto separate columns containing 1 ml wheat germ agglutinin (WGA)Sepharose. Each column was washed and then eluted as described (8). GVIA-binding was determined by gelfiltration as described for solubilization. DHP-binding was measured by liquid scintillation counting. 948

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Our

BIOCHEMICAL

AND

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RESEARCH

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studies

with the membranes of adult chicken ing sites with a Bmax of pmol/mg for DHP and GVIA, 0.536 + 0.271 pM for DHP equal

amount

using

juvenil

VDCC blockers DHP and GVIA using brain revealed a rather equal amount of bind0.403 + 0.11 pmol/mg or 0.315 + 0.124 repectively. The k, was 180 + 59 pM or and GVIA, respectively (Fig. 1). This of binding sites is in contrast to previous reports brain tissues, which revealed 5-fold more GVIA- than

DHP-binding

sites

precipitate

any

(6,

9).

However,

GVIA-binding

Hayakawa

sites

from

et

al.

adult

rabbit

brains using a L-type specific antibody. This and GVIA-binding occurs on different targets, were

used. Taken

during i)

their

both

types

led

to

of

we

binding

sites

a different

different

using

chicken

in

with

brains

1.25

present

from

adult

% digitonin. in

membranes

were

chicken,

could

be recoverd

prelabeled

with

of

the

bovine

that because

two

types

of

work (12, 13), our investigations

whereas

15 % of

not

conclude

other

the

the

after

(15). binding membranes

DHP-binding

solubilization,

[3H]PN 200-110

prior

ii)

investiga-

root ganglion two types of

we solubilized

Approximitly

membranes

can

by previous brain, since

used whole brain tissue (14) or dorsal In order to further characterize the

and

may change

ratio

cerebrum,

could

indicates that DHPwhen adult tissues

together,

which is supported distribution in

sites, performed

sites

if

observations

expression

binding

their were tions

these

development

(12)

sites only

to solubili-

b 300

500

PN 200.110

Figure 1. GVIA-binding

Concentration

-r

1000

(PM)

GVIA

specific PN 200-llOand Chicken brain membranes were incubated with the indicated concentrations of [3H]PN ZOO-110 (panel a) and [ 125J]GVIA (panel b). Bound (B) and free (F) label was separated by rapid filtration (post label method). In both cases nonspecific binding was less than 20 % of the total binding. The values are the mean + standard deviation of four independent experiments. The inserted blot shows the transformation of the data according to Scatchard (20). to

chicken

dependence

brain

of

(PM)

membranes.

949

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1992

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B (Imdlmg protein) 300

600

PN 200-110

COMMUNICATIONS

B (Imolhg protein)

900

0

1000

500

(PM)

PN 200-110

1500

(PM)

Ficfure 2. Comparison of specific DHP binding to membraneand solubiliaed fractions of chicken brain. Membranes were labeled with the indicated amounts of [3H]PN 200-110, bound and free label was separated by centrifugation (prelabel method). The amount of bound DHP was determined before (panel a) and after solubilization Unspecific binding was less than 20 % in membranes and (panel b). 50 % in solubilized brain fractions. The inserted blot shows the transformation of the data according to Scatchard (20).

zation

(Fig.2).

This

solubilization

of

and heart solubilization loss from

compared

to

the

same

the

DHP-binding to

in

contrast

sites

type

of

binding

(17)

the

be due

However, it is not unlikely caused by an interaction of

a corresponding

observed

(12, the

DHP-binding sites to solubilization derived

supports

detergent

interference that a lipid

that

both

from

further graphic techniques sites from muscle

with

muscle non-DHPcurrents

sensitivity subunit

inter-

this detergent sensitiviwith the calcium channel GVIA-binding sites coshown), indicating that sensitivity to the so-

binding

Two populations of 19), one of them contains DHP-binding site.

For

(2)

(8) reported that led also to a great

sites

(18). In contrast to the DHP-binding sites, uld be successful solubilized (data not both types of binding sites have different

described represents

successful muscle

investigations demonstrated that L-type VDCC modulate the calcium

could

which targets.

the

skeletal

This indicated that a higher sensitivity

action. ty is

lubilization, on different

to

from

Curtis and Catterall membranes from rat

sites. have

Since previous subunits of the and

is

DHP-binding

However, of brain

of DHP-binding neuronal tissues

tissue. binding (16)

(3).

observation

the

sites

are

located

N-type VDCC have been the al-subunit which

biochemical characterization we used chromatowhich were well established for DHP-binding tissues (2, 3). Solubilized membranes were loaded onto 1 ml DEAE-Sepharose column. After washing the column both types could be eluted at nearly the same salt concentration 950

Vol.

182, No. 2, 1992

-flow

BIOCHEMICAL

through

100

AND BIOPHYSICAL

200

300 NaCl

riuure

from

400

RESEARCH COMMUNICATIONS

500

1000

(mM)

3. Anion exchange chromatography of DliP- and GVIA-receptors chicken brain. Solubilized and membranes prelabeled with

[3H]PN 200-110 or [125 JIGVIA were loaded onto a DEAE-column and eluted with increasing concentrations of NaCl. The amount DHP- and GVIA-binding sites applied to the column was set to 100 %.

(0.2 M NaCl) with similar recoveries, indicating that both types have similar binding characteristics upon ion exchange chromatography (Fig. 3). In contrast to this observation, a comparable investigation using a glycoprotein affinity column revealed that both types of binding sites have different binding properties. Different amounts of both binding sites could be bound to the wheat germ agglutinin (WGA)-column. Most of the GVIA-binding sites (73.9 % of the loaded binding sites) bound to the lectin, whereas 62.3 % of the loaded DHP-binding sites were found in the flow through fraction. Since both binding sites were loaded under similar conditions, the different partitioning in the fractions of the glycoprotein affinity chromatography could be mainly due to a corresponding different degree of glycosylation, which again supports that both binding sites may be a different target. In summary our results support that the ligands GVIA and DHP have different binding sites, which is in agreement to corresponding investigations using brain tissues from adult rabbit and bovine (12, 19). In particular, we observed different sensitivity to solubilization and a different partitioning upon chromatography with a glycoprotein affinity column. In addition we determined rather equal amounts of the two types of binding sites indicating that either expression or distribution of the two types of binding sites

have

changed

during

development. 951

We therefore

conclude

that

Vol.

182, No. 2, 1992

the both VDCC-types of neuronal tissue.

BIOCHEMICAL

may play

AND BIOPHYSICAL

a so far

unknown

RESEARCH COMMUNICATIONS

role

in

development

REFERENCES 1) McCleskey, E. W., Fox, A. P., Feldman, D. H., Cruz, L. J., Olivera, B. M., Tsien, R. W. and Yoshikami, D. (1987) Proc. Natl. Acad. Sci. USA 84, 4327-4331. F. (1986) Eur. J. 2) Flockerzi, V., Oeken, H. J. and Hofmann, Biochem. 161, 217-224. 3) Mikamie, A. M., Imoto, K., Tanabe, T., Niidome, T., Mori, Y. , Takeshima, H., Marumiya, S. and Numa, S. (1989) Nature 340, 230-233. 4) Campbell, K. P., Leung, A. T. and Sharp, A. H. (1988) TINS 11, 425-430. 5) Nowycky, M. C., Fox, A. P. and Tsien, R. W. (1985) Nature 316, 440-443. 6) Takahshi, M. and Fujimoto, Y. (1989) Biochem. Biophys. Res. Commun. 163, 1182-1188. 7) Glossmann, H. and Ferry, D. R. (1985) Methods in Enzymol. 109, 513-550. 8) Curtis, B. M. and Catterall, W. A. (1983) J. Biol. Chem. 258, 7280-7283. 9) Barhanin, J., Schmid, A. and Lazdunski, M. (1988) Biochem. Biophys. Res. Commun. 150, 1051-1062. 10) Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 11) Yamaguchi, T., Saisu, H., Mitsui, H. and Abe, T. (1988) J Biol. Chem. 263, 9491-9498. 12) Hayakawa, N., Morita, T., Yamaguchi, T., Mitsui, H., Mori,K. J., Saisu, H. and Abe T. (1990) Biochem. Biophys. Res. Commun. 173, 483-489. 13) Aosaki, T. and Kasai, H. (1989) Eur. J. Physiol. 414, 150-m. 14) Cruz, L. J. and Olivera, B. M. (1986) J. Bio. Chem. 261, 623+ 6233. 15) Feldman, D. H., Olivera, B. M. and Yoshikamie D. (1987) Febs Letters 214, 295-300. 16) Singer, D., Biel, M., Lotan, I., Flockerzi, V., Hofmann, F. and Dascal, N. (1991) 253, 1553-1557. 17) Woscholski, R. and Marme, D. (1991) Cell. Signalling, in pzees 18) Glossmann, H. and Ferry, D. R. (1983) Nauny-Schmiedebergs Arch. Pharmacol. 323, 279-291. 19) Ahlijanian, K., M. Striessnig, J. and Catterall, W. A. (1991) J. Biol. Chem. 266, 20192-20197. 20) Scatchard, G. (1949) Annu. N. Y. Acad. Sci. 51, 660-672.

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