- Email: [email protected]
The molecular target size of brain TBPS binding sites
European Journal of Pharmacology, 96 (1983) 321-322
321
Elsevier
Rapid communication THE MOLECULAR TARGET SIZE OF BRAIN TBPS BINDING SITES MOGENS NIELSEN 1., and CLAUS BRAESTRUP 1.2 /Sct. Hans Hospital, Psychopharmacological Research Laboratory, DK-4000 Roskilde, Denmark, and 2 A / S Ferrosan, Research Division, Sydmarken 5, DK-2860 Soeborg, Denmark
Received 7 November 1983, accepted 18 November 1983
The G A B A / b e n z o d i a z e p i n e receptor chloride channel complex appears to be a macromolecular complex of m e m b r a n e b o u n d proteins. The G A B A receptor function has been characterized by neurophysiological and receptor binding techniques. The benzodiazepine receptor was originally characterized by high affinity binding techniques and was later shown to be in a tight allosteric coupling to the G A B A receptor. The molecular weight of the benzodiazepine receptor m o n o m e r is ca 50 000 daltons as determined by SDS-gel electrophoresis and high energy irradiation techniques (Mibhler et al., 1980; Paul et al., 1981). The chloride gating mechanism is less well characterized. Electrophysiological studies show that the G A B A and benzodiazepine receptor exert a concerted regulatory function on the chloride channels. Direct demonstrations of the chloride gating complex or parts thereof have been achieved with [3H]dihydropicrotoxinin and recently by [35S]TBPS ([35S]tbutylbicyclophosphorothionate) as radioligands (Squires et al., 1983). In the present study we have investigated the target size of the [35S]TBPS binding complex by the radiation inactivation technique ( K e m p n e r and Schlegel, 1979). After various doses of radiation, frozen rat cortex tissue was homogenized in 2 × 10 vol of 50 m M Tris, citrate buffer p H 7.1, by an Ultra-Turrax homogenizer for 10 s at 0 ° C followed by centrifugation at 30000 × g for 15 min. T h e pellet was washed thrice by homogenization and centrifugation as before and the final pellet was resuspended in 100 vol Tris citrate buffer (50 m M , p H 7.1) containing 1000 m M NaC1. Aliquots of 0.5 ml were incubated for 3 h at 25°C with 0.9 n M [35S]TBPS ( N E N , 93 C i / m m o l ) in the absence 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
and presence of 30 # M picrotoxinin for determination of nonspecific binding. Free and b o u n d radioactivity was separated by filtration through What-
~,,..
9
[all]
F lunitrazepam
0
E
Q. 0
.E "o .E
8
.~_ 0 r,o
~
[35S] TBPS O,k %. k =0.183 Mrad "1 ~(~
7
0
I
I
I
I
2
4
6
8
I
Dose, Fig.
1.
I
I
I
I
I
10 12 14 16 18 20 Mrad
Radiation inactivation of [35S]TBPS (O) and
[3H]flunitrazepam binding sites (zx), which were investigated in parallel (NEN 76.9 Ci/mmol, 3 riM, 2 mg tissue/ml, Tris citrate buffer 50 mM pH 7.1 containing 1000 mM NaCl, 0°C, blank 3 #M midazolam). For calibration the radiation inactivation constant, k Mrad-1, was estimated for 7 enzymes with known molecular weight (Mw daltons): yeast alcohol dehydrogenase (148000), k = 0.202; E. Coil fl-galactosidase (116248), k = 0.157; pyruvate kinase (114000), k = 0.152; horse liver alcohol dehydrogenase (72000), k = 0.095; lactate dehydrogenase (70000), k = 0.108; cholinesterase (70.000), k = 0.087; glutamic acid decarboxylase (67000), k = 0.098. A proportional factor of 730000 daltons. Mrad was determined as the mean for all enzymes.
322
man G F / C glass filters followed by washing with 3 × 5 ml icecold buffer. This binding procedure has been optimized. The K D for [35S]TBPS was 12 nM, the Bmax value was 149 pmol/g, which is very close to the value of 130 p m o l / g obtained in the same experiment for [3H]flunitrazepam. IBTBO, picrotoxin, anisatin, etomidate and SQ 20009 are good inhibitors of [35S]TBPS binding under the conditions described here. Whole frozen rat cortices were exposed to ionizing radiation with high energy electrons using the 10 MeV linear accelerator at Riso, Denmark. The dose of radiation was determined using calibrated thermo dosimeters (water). The samples were cooled (ca. - 1 0 ° C ) during radiation which was delivered in runs of 0.5-2 Mrad. Between runs, samples were cooled to - 1 5 ° C for at least 2 min to ensure that they remained completely frozen during the whole irradiation process. Fig. 1 shows that the decay of [35S]TBPS binding proceeds in a monoexponential fashion; the negative slope, which represents the radiation inactivation constant k, is 0.183 Mrad -1, corresponding to a molecular weight of 730 000 × 0.183 = 134000 daltons (see fig. 1 legend). Under the same conditions the molecular size of the [3H]flunitrazepam binding site was determined to be 54000 daltons (k = 0.0740, fig. 1) in agreement with other studies. The target size of [3H]muscimol binding sites in these conditions is 53 000 daltons (Nielsen and Braestrup, in preparation). The disappearance of [35S]TBPS binding sites corresponds to a loss of binding sites rather than a change in affinity, because the ICs0 of unlabelled
TBPS as inhibitor on [35S]TBPS was unchanged from 0-10 Mrad (data not shown). These results show that the target size of [35S]TBPS (134000 daltons) is different from both the benzodiazepine receptor monomer and the GABA receptor (both ca. 50 000 daltons) and also from any known multimer benzodiazepine receptor complex, (such as ca. 200000 daltons (Chang et al., 1981)). The results suggest that the chloride gating mechanism contains proteins distinct from the GABA and the benzodiazepine receptor proteins. It should not be overlooked, however, that the size of 134000 daltons is not necessarily one single protein. Several molecular entities may be included in this value.
References Chang, L.-R., E.A. Barnard, M.M.S. Lo and J.O. Dolly, 1981, Molecular sizes of benzodiazepine receptors and the interacting GABA receptors in the membrane are identical, FEBS Lett. 126, 309. Kempner, E.S. and W. Schlegel, 1979, Size determination of enzymes by radiation inactivation, Anal. Biochem. 92, 2. M6hler, H., M.K. Battersby and J.G. Richards, 1980, Benzodiazepine receptor protein identified and visualized in brain tissue by a photoaffinity label, Proc. Natl. Acad. Sci. U.S.A. 77, 1666. Paul, S.M., E.S. Kempner and P. Skolnick, 1981, In situ molecular weight determination of brain and peripheral benzodiazepine binding sites, Europe,an J. Pharmacol. 76, 465. Squires, R.F., J.E. Casida, M. Richardson and E. Saederup, 1983, [35S]t-butylbicyclophosphothionate binds with high affinity to brain specific sites coupled to y-aminobutyric acid-A and ion recognition sites, Mol. Pharmacol. 23, 326.
321
Elsevier
Rapid communication THE MOLECULAR TARGET SIZE OF BRAIN TBPS BINDING SITES MOGENS NIELSEN 1., and CLAUS BRAESTRUP 1.2 /Sct. Hans Hospital, Psychopharmacological Research Laboratory, DK-4000 Roskilde, Denmark, and 2 A / S Ferrosan, Research Division, Sydmarken 5, DK-2860 Soeborg, Denmark
Received 7 November 1983, accepted 18 November 1983
The G A B A / b e n z o d i a z e p i n e receptor chloride channel complex appears to be a macromolecular complex of m e m b r a n e b o u n d proteins. The G A B A receptor function has been characterized by neurophysiological and receptor binding techniques. The benzodiazepine receptor was originally characterized by high affinity binding techniques and was later shown to be in a tight allosteric coupling to the G A B A receptor. The molecular weight of the benzodiazepine receptor m o n o m e r is ca 50 000 daltons as determined by SDS-gel electrophoresis and high energy irradiation techniques (Mibhler et al., 1980; Paul et al., 1981). The chloride gating mechanism is less well characterized. Electrophysiological studies show that the G A B A and benzodiazepine receptor exert a concerted regulatory function on the chloride channels. Direct demonstrations of the chloride gating complex or parts thereof have been achieved with [3H]dihydropicrotoxinin and recently by [35S]TBPS ([35S]tbutylbicyclophosphorothionate) as radioligands (Squires et al., 1983). In the present study we have investigated the target size of the [35S]TBPS binding complex by the radiation inactivation technique ( K e m p n e r and Schlegel, 1979). After various doses of radiation, frozen rat cortex tissue was homogenized in 2 × 10 vol of 50 m M Tris, citrate buffer p H 7.1, by an Ultra-Turrax homogenizer for 10 s at 0 ° C followed by centrifugation at 30000 × g for 15 min. T h e pellet was washed thrice by homogenization and centrifugation as before and the final pellet was resuspended in 100 vol Tris citrate buffer (50 m M , p H 7.1) containing 1000 m M NaC1. Aliquots of 0.5 ml were incubated for 3 h at 25°C with 0.9 n M [35S]TBPS ( N E N , 93 C i / m m o l ) in the absence 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.
and presence of 30 # M picrotoxinin for determination of nonspecific binding. Free and b o u n d radioactivity was separated by filtration through What-
~,,..
9
[all]
F lunitrazepam
0
E
Q. 0
.E "o .E
8
.~_ 0 r,o
~
[35S] TBPS O,k %. k =0.183 Mrad "1 ~(~
7
0
I
I
I
I
2
4
6
8
I
Dose, Fig.
1.
I
I
I
I
I
10 12 14 16 18 20 Mrad
Radiation inactivation of [35S]TBPS (O) and
[3H]flunitrazepam binding sites (zx), which were investigated in parallel (NEN 76.9 Ci/mmol, 3 riM, 2 mg tissue/ml, Tris citrate buffer 50 mM pH 7.1 containing 1000 mM NaCl, 0°C, blank 3 #M midazolam). For calibration the radiation inactivation constant, k Mrad-1, was estimated for 7 enzymes with known molecular weight (Mw daltons): yeast alcohol dehydrogenase (148000), k = 0.202; E. Coil fl-galactosidase (116248), k = 0.157; pyruvate kinase (114000), k = 0.152; horse liver alcohol dehydrogenase (72000), k = 0.095; lactate dehydrogenase (70000), k = 0.108; cholinesterase (70.000), k = 0.087; glutamic acid decarboxylase (67000), k = 0.098. A proportional factor of 730000 daltons. Mrad was determined as the mean for all enzymes.
322
man G F / C glass filters followed by washing with 3 × 5 ml icecold buffer. This binding procedure has been optimized. The K D for [35S]TBPS was 12 nM, the Bmax value was 149 pmol/g, which is very close to the value of 130 p m o l / g obtained in the same experiment for [3H]flunitrazepam. IBTBO, picrotoxin, anisatin, etomidate and SQ 20009 are good inhibitors of [35S]TBPS binding under the conditions described here. Whole frozen rat cortices were exposed to ionizing radiation with high energy electrons using the 10 MeV linear accelerator at Riso, Denmark. The dose of radiation was determined using calibrated thermo dosimeters (water). The samples were cooled (ca. - 1 0 ° C ) during radiation which was delivered in runs of 0.5-2 Mrad. Between runs, samples were cooled to - 1 5 ° C for at least 2 min to ensure that they remained completely frozen during the whole irradiation process. Fig. 1 shows that the decay of [35S]TBPS binding proceeds in a monoexponential fashion; the negative slope, which represents the radiation inactivation constant k, is 0.183 Mrad -1, corresponding to a molecular weight of 730 000 × 0.183 = 134000 daltons (see fig. 1 legend). Under the same conditions the molecular size of the [3H]flunitrazepam binding site was determined to be 54000 daltons (k = 0.0740, fig. 1) in agreement with other studies. The target size of [3H]muscimol binding sites in these conditions is 53 000 daltons (Nielsen and Braestrup, in preparation). The disappearance of [35S]TBPS binding sites corresponds to a loss of binding sites rather than a change in affinity, because the ICs0 of unlabelled
TBPS as inhibitor on [35S]TBPS was unchanged from 0-10 Mrad (data not shown). These results show that the target size of [35S]TBPS (134000 daltons) is different from both the benzodiazepine receptor monomer and the GABA receptor (both ca. 50 000 daltons) and also from any known multimer benzodiazepine receptor complex, (such as ca. 200000 daltons (Chang et al., 1981)). The results suggest that the chloride gating mechanism contains proteins distinct from the GABA and the benzodiazepine receptor proteins. It should not be overlooked, however, that the size of 134000 daltons is not necessarily one single protein. Several molecular entities may be included in this value.
References Chang, L.-R., E.A. Barnard, M.M.S. Lo and J.O. Dolly, 1981, Molecular sizes of benzodiazepine receptors and the interacting GABA receptors in the membrane are identical, FEBS Lett. 126, 309. Kempner, E.S. and W. Schlegel, 1979, Size determination of enzymes by radiation inactivation, Anal. Biochem. 92, 2. M6hler, H., M.K. Battersby and J.G. Richards, 1980, Benzodiazepine receptor protein identified and visualized in brain tissue by a photoaffinity label, Proc. Natl. Acad. Sci. U.S.A. 77, 1666. Paul, S.M., E.S. Kempner and P. Skolnick, 1981, In situ molecular weight determination of brain and peripheral benzodiazepine binding sites, Europe,an J. Pharmacol. 76, 465. Squires, R.F., J.E. Casida, M. Richardson and E. Saederup, 1983, [35S]t-butylbicyclophosphothionate binds with high affinity to brain specific sites coupled to y-aminobutyric acid-A and ion recognition sites, Mol. Pharmacol. 23, 326.