modification inhibits BZD binding (Herblin and. Mechem, 1984). Scatchard analysis of (3H]FNZM binding to homogenates of 20-day embryonic chick brain sug-.
European JOl/mal of PharmacologJ', 110 (1985) 171-180 Elsevier
171
BENZODIAZEPINE RECEPTOR PHOTOAFFINITY LABELING: CORRELATION OF FUNCTION WITH BINDING T.T. GIBBS,
c.y. CHAN, C.M.
CZAJKOWSKI and D.H. FARB •
Department of Anatomy and Cell Biology, Downstale Medical Cenler, The State University of New York. Brooklyn, NY 1/203, U.S.A.
Received 12 September 1984. revised MS received 11 December 1984, accepled 2 January 1985
T.T. GIBBS, c.Y. CHAN, C.M. CZAJKOWSKI and D.H. FARB. Benzodiazepine receptor photoaffinity labeling: correlation of function with binding. European J. Pharmacol. 110 (1985) 171-180.
Exhaustive photoaffinity coupling of f1unitrazepam to living spinal cord neurons reduced the capacity of benzodi· azepines to potentiate the electrophysiologically measured GADA response. In qualitative agreement with reversible binding data the dose-response curve for enhancement of the GADA response by benzodiazepines was shifted to the right. indicating thaI Ihe remaining reversible benzodiazepine binding sites have lower affinity for benzodiazepines. Photoaffinity labeling did not reduce inhibition of the GADA response by p.carbolines and there was only a small decrease in p-carboline binding. In both control and photoaffinity-Iabeled cultures, the inhibitory effect of p·carbolines on the GABA response was reversed in the presence of excess benzodiazepine. The results indicate that the effects of photoaffinity labeling are confined to the BZD recognition site, and that coupling between benzodiazepine receptors and GABA receptors remains intact. Benzodiazepine
p-Carboline
Photoaffinity labeling
1. Introduction
.-
.~
It is becoming increasingly apparent that the GABA receptor is an important target for a wide variety of pharmacological agents. Prominent among these arc the benzodiazepines (BZDs), which have long been used in clinical medicine for their anxiolytic, anticonvulsant and muscle relaxant properties. Studies of binding of radiolabeled BZDs to brain membrane homogenates have led to the characterization of a variety of non-BZD ligands that apparently compete with BZDs for binding (Braestrup et aI., 1980, 1982a; O'Brien et aI., 1981). A number of these, including the /l-carboline esters methyl-/l-carboline-3-carboxylate (BCCM) and methyl.6,7·dimethoxy-4ethyl-,B-carboline-3-carboxylate (DMCM) have effects in vivo which are qualitatively the inverse of the BZDs; that is, they promote or induce convul• To whom all correspondence should be addressed: Dept. of Anatomy & Cell Biology. Downstale Medical Center, Box 5. 450 Clarkson Ave, Brooklyn. NY 11203. U.S.A. 0014·2999/85/S03.30
of:
1985 Elsevier Science Publishers B.V.
GADA
sive states or increase anxiety (Oakley and Jones, 1980; Braestrup et al., 1982b; Schweri et al., 1982; Jensen et al., 1983). Neither BZDs nor /l-carbolines induce an electrophysiological response when applied to spinal cord neurons in dissociated cell culture; instead, these drugs alter the magnitude of the conductance increase. gGABA, that occurs in response to application of GABA. When neurons are pretreated with classical BZDs, such as flunitrazepam (FNZM), clonazepam, or chlordiazepoxide (CDPX). then challenged with GABA, the conductance response to submaximal GABA concentrations is increased over that observed in the absence of BZD treatment (Choi et al., 1977, 1981; MacDonald and Barker, 1978; White et aI., 1981; Chan et al., 1983). In contrast, the ,B-carbolines DeCM and DMCM have been shown to decrease gGABA (Farb et al., 1984). The BZD receptor may be photoaffinity labeled by exposure to ultraviolet light in the presence of the benzodiazepine FNZM. By this method it is possible to irreversibly link (3H]FNZM to BZD
172
receptors in homogenates of rat (Mohler et al.. 1980) or chick brain, or in cultures of embryonic chick brain or spinal cord neurons (Chan et aI., 1983). Maximum photoaffinity labeling is typically about 25% of maximum reversible binding (Mohler et aI., 1980). Arter exhaustive photoaffinity labeling with nonradioactive. FNZM, the remaining reversible binding sites continue to bind BZDs, but with lower affinity (Karobath and Supavilai, 1982; Mohler, 1982; Thomas and Tallman, 1983). Notably, the inhibitory effect of photoaffinity labeling is greater for BZD binding than for f3carboline binding (Hirsch, 1982; Gee and Yamamura, 1982; Karobath and Supavilai, 1982; Thomas and Tallman, 1983). It is unclear why only some sites can be photoaffinity labeled with FNZM, nor is it known why affinity of BZD binding is lowered for those sites which do not become irreversibly labeled. Thus, it is impossible to predict, from binding results alone, the effect of photoaffinity labeling upon BZD receptor function. In particular, the question of whether photoaffinity-modified BZD receptors retain their capacity to enhance the e1ectrophysiological GABA response has not previously been addressed. We report here a correlative study of the effects of FNZM-photoaffinity linkage upon the ligand-binding and functional characteristics of the BZD receptor. Exhaustive photoaffinity labeling of BZD receptors in brain membranes lowered the affinity of the remaining reversible BZD binding site for BZDs, but had little effect on the binding of f3-carbolines. When living neurons were photoaffinity labeled, enhancement of the GABA response by BZDs was reduced and the dose-response curve for enhancement was shifted to the right, consistent with lower affinity binding of DZDs to the BZD recognition site. f3-Carboline inhibition of the GADA response was unaffected by photoaffinity labeling. The results indicate that photoaffinity labeling alters the characteristics of the DZD recognition site, but leaves intact the coupling between DZD binding and enhancement of the GABA response.
2. Materials and methods
2. J. Chemicals The benzodiazepine drugs flunitrazepam (FNZM), chlordiazepoxide (CDPX), and c1onazepam, and the f3-carboline DCCM were gifts of Dr. W. Scott of F. Hoffman-La Roche (Nutley, NJ). DMCM was a gift of Dr. Claus Braestrup of A/S Ferrosan (Soeborg, Denmark). All other chemicals were obtained from commercial sourses. 2.2. Cell culture
Spinal cord cultures were prepared as previously described (Farb et aI., 1979). Spinal cords were dissected from 7-day chick embryos. Sensory ganglia and meninges were removed and the cords were minced into small fragments. The tissue fragments were incubated with 0.075% trypsin (GIBCO) for 5 min at 37°C in Puck's DIG (a Ca2+- and Mg 2 + -free saline), then the tissue was separated from the trypsin-containing saline by centrifugation and resuspended in complete medium (Eagle's MEM, supplemented with 2.4 mM glutamine, 18.3 mM glucose, 10% heat-inactivated horse serum. 5% chick embryo extract. 50 units/ml penicillin and 50 JLg/mJ streptomycin). The tissue fragments were triturated through a fire-polished Pasteur pipette and plated on collagen-coated plastic 35 mm tissue culture dishes at a concentration of ca. 400 cells/mm2• Cultures were maintained at 37°C in an atmosphere of 5% CO2·95% air. Cytosine arabinoside (111M) was added after 2-3 days to control the proliferation of nonneuronaI cells. Cultures were fed with fresh medium I day later, and thereafter at 2-3 day intervals as needed. ElectrophysiologicaJ experiments were generally performed 3-6 weeks postplating. 2.3. Membrane homogena/es
.
i
Binding experiments were performed upon P2 membrane homogenates of whole brains dissected from 20-day chick embryos. Ideally, it would be preferable to measure binding in the same preparation (cultures of 7-day embryonic spinal cord)
..
173
~
as that used for electrophysiology. This approach was not taken because the yield of BZD binding sites from 7-day embryos was much lower than from 20.day embyos. and because binding to 7-day tissue was complicated by the presence of large numbers of low-affinity BZD binding sites which apparently do not contribute to enhancement of the GADA response. In contrast, homogenates of 20-day brain exhibit only high-affinity BZD binding sites, which we have previously shown to correa late well with enhancement of the GABA response in 7-day spinal cord cultures (Chan et al.• 1983). Whole brains were removed. homogenized (10 volumes. 0.32 M sucrose, O°C) and centrifuged (5 min, 1000 x g, 4°C). The supernatant was centri· fuged (20 min. 30000 x g) to yield a P2 pellet which was resuspended (3 mg protein/ml) in PBSS (116.4 mM NaCI. 5.4 mM KCI, 1 mM CaCI 2 • 0.8 mM MgS04 • 22 mM glucose. 11 mM NaH 2 P04 • pH 7.4). washed at least twice by centrifugation and stored at - 70°C until used. Binding experiments were performed at O°c. Membranes were thawed and incubated 30 min with (3H]FNZM or (3H]BCCM. Radioactivity reIJff1!"'\ tained on Whatman GF/B glass fiber filters after { four 5 ml washes was determined by liquid scintillation counting. Nonspecific binding was determined in the presence of 1 mM flurazepam (for 13 HlFNZM binding) or 10 11M BCCM (for (3H]BCCM binding). 1.4. Electrophysiology
~
Electrophysiological experiments were performed in 35 mm tissue culture dishes on the stage of an inverted phase-contrast microscope. Cultures were perfused (70 mljh) with an oxygenated mixture of Earle's DSS. pH 7.2-7.4, supplemented with 0.2% horse serum, 3.2 mM CaCI 2 and 16 mM glucose and maintained at 36-37°C. Membrane potential was measured by standard intracellular recording techniques using a microelectrode (R = 60-100 MU) filled with 0.5 M potassium acetate. Membrane conductance was determined by Ohm's law from the amplitude of the voltage response elicited by constant current pulses (0.1 nA, 100 ms, 2 Hz) injected across the membrane by means of a bridge circuit. Only those neurons that yielded
stable recordings of membrane potential (at least - 60 mY) and input resistance and linear currentvoltage relationsmps over a wide range were selected for study. GABA and benzodiazepines were dissolved in BSS and applied to single neurons by pressure ejection from 7-barrel blunt tip (5-7 11m barrel) micropipets. Water-insoluble compounds were first dissolved in dimethyl sulfoxide (DMSO). then diluted in recording medium to a final DMSO concentration of 0.1 %, which control experiments showed to be without effect when applied to spinal cord neurons. Under the experimental conditions. pressure ejection effectively replaces the bathing solution surrounding the target neuron with the contents of the pressure pipet. Thus. the neuronal membrane is exposed, during pressure ejection, to the same drug concentration as that in the pipet (Choi et aI., 1981). Conductance responses to a given concentration of GABA agreed to witmn a 10% tolerance. regardless of whether GABA was applied by pressure ejection or in the batmng solution. The GABA response was defined as the increase in membrane conductance in response to GABA application (gGABA) according to the equation gGABA = gobs - gm where gm is the rest· ing membrane conductance measured before GABA application and gobs is the peak membrane conductance during the GADA pulse. The pressure pulse was terminated when a maximal response was observed. The position of the pipet tip was not critical when within 50 11m of neuronal somata. No spurious membrane responses due to leakage of GABA were observed in the absence of a pressure pulse. To minimize the risk of crosscontamination between barrels, the pressure pipet was lifted away from the immediate vicinity of the neuron during the intervals between pressure pulses. Control experiments verified that there was no cross-contamination. since a buffer blank in a barrel adjacent to one containing 10 mM GABA failed to elicit a conductance increase when applied to neuronal perikarya. GABA pulses were applied in ascending order of concentration. To avoid desensitization. each GABA pulse was fol· lowed by a 3-5 min recovery period.
174
2.5. Determination of dose-response curves for pote1lliation of gGABA To determine dose-response curves for BZO potentiation of GABA chemosensitivity. a fixed GABA concentration was selected for the test pulse and potentiation measured after application of BZD. GABA responses were measured before (gGABA) and after (gGABA') application of BZD. Enhancement of the GABA response is conveniently expressed in terms of 0:: := (gGABA'/gGABA - 1) x 100 where 0:: may be positive. zero. or negative when gGABA is potentiated, unaltered, or inhibited, respectively. The dose-response relationship for enhancement or inhibition of GABA was determined individuaJ)y for each neuron tested using 4 to 6 concentrations of benzodiazepine or ,8-carboline, applied with a multibarrel pressure pipet. The maximal enhancement amax and ECso for each neuron was estimated by Eadie-Hofstee analysis. To obtain reproducible dose-response curves for benzodiazepines, it was important to select a test concentration of GABA which was large enough to produce a clear change in membrane conductance, but not so large that the voltage deflection elicited by the constant current pulses became too small to measure. In order to detect changes in the maximum enhancement, O::max' it was also necessary that the test concentration be low enough so that the measured gGABA was well below its maximum value both in the presence and absence of BZDs. Unless otherwise specified, the concentration of GABA was 2 JLM, which elicited a control response less than 5% of the maximal conductance gGABA max obtainable at high [GADA]. Even at this low concentration of GABA the combination of a large resting conductance and a large gGABA led to difficulties in balancing the bridge circuit with some neurons. Results were discarded if an unambiguous bridge balance could not be obtained.
2.6. Photo;nactivation of BZD binding The term 'photoinactivation' will be used to refer to exhaustive photoaffinity labeling of BZD receptors in membrane homogenates or spinal cord
cultures with nonradioactive FNZM. To photoinactivate primary cultures of chick spinal cord neurons. the cultures were washed twice with PBSS. incubated with 100 nM unlabeled FNZM (4°C. 30 min), and irradiated (20 min, 4°C) with long wavelength ultraviolet light (General Electric F40BLB bulb, I cm). These conditions were found to be sufficient to produce maximal irreversible binding of FNZM, as well as maximal inhibition of subsequent reversible binding. Cells were extensively washed with PBSS to remove trapped and reversibly bound FNZM. Except when otherwise specified. control cultures were incubated with FNZM, but were not exposed to UV light. Photoinactivation of homogenates of lO-day chick brain was performed similarly (Chan et al.. 1983). After photoinactivation membranes were washed by centrifugation 4 times, to eliminate free and reversibly bound FNZM. Controls were incubated with FNZM and washed as described above. but were not exposed to UV light.
~ .
3. Results
3.1. [JH]FNZM binding: effect of photoinactivat;on When brain membrane homogenates or brain or spinal cord cell cultures were UV-irradiated in the presence of [3HIFNZM, up to 25% of the reversible BZD binding sites became irreversibly labeled. Reversible binding of [J HjFNZM to the remaining sites was inhibited. To examine this effect in detail, membranes were photoaffinity labeled ('photoinactivated') with FNZM (100 nM, 20 min), washed to remove free and reversibly bound FNZM. then assayed to determine the concentration dependence of benzodiazepine binding. Binding of [3HjFNZM to control membranes was hyperbolic, consistent with a single class of sites characterized by a K d of 5·10 nM (fig. 1). After photoinactivation, reversible binding of (3HjFNZM could still be demonstrated, but the binding curve was shifted to the right (ECso > 100 nM) indicating lower affinity. The decrease in binding affinity for FNZM is primarily responsible for the large decrease observed when reversible binding is measured at a fixed concentration of
""J
"
175
3
20C-:~O"
A
B
0
o
O~ ,to
=.. 2
Ol-
e
e
-0
0
10
~ ~t
o~~
all/ l'\: . ., ~. .. o·
Co.
"'00
e e
al ~
l~.
I
I
E
o
Co.
0/ ,/
o
...
0·0 ~_ ...JLL!lIl:"'::....L.."
-9
-8
-------'
•
c
•
..I--_ _---'
-6
-1
log [FI unitroz epomJ
I
0'---,
M
Fig. I. Binding: effect of photoinaclivation. A: saturation binding of (3H]FNZM to control and photoinactivaled 20-day brain P2 membranes. Specific activity of 13HIFNZM was adjusted by addition of unlabeled FNZM over the range }2-83 X 10 3 Cijmmo\. Circles: conlrol membranes (exposed 10 FNZM bUI not UV); squares: photoinaclivated membranes. Band C: same dala as A. Scalchard plots.
(3H]FNZM. Whereas Scatchard plots of binding to control membranes were linear, indicating that r'H]FNZM binding sites are homogeneous in affinity, Scatchard plots (fig. IB,C) of binding to photoinactivated membranes were curved, suggesting that photoinactivated sites are heterogeneous with respect to affinity for [3H]FNZM. The results suggest that the reduction in affinity is nonuniform, such that some [3H]FNZM binding sites are more affected than others. If the photoaffinity labeling procedure links FNZM to its usual binding site rather than to some other part of the BZD receptor/GABA receptor complex, then the Bmax for reversible binding to photoinactivated membranes should be reduced by about 25%, corresponding to the fraction of sites which irreversibly bind FNZM. While Bmax for photoinactivated membranes could not be determined directly due to increased nonspecific binding at high (3 H]FNZM concentrations, reversible binding to photoinactivated membranes was about 60% of control at 700 nM (3H]FNZM, approaching the expected value of 75%. .'
3.2. Electrophysiology: effect of photoinactivation on BZD receptor function
To examine the functional consequences of photoinactivation, spinal cord cultures were photo-
inactivated, washed extensively to remove free and reversibly bound FNZM, and assayed for enhancement of gGABA by CDPX. Photoinactivation had no detectable effect on the morphology or resting potentials of spinal cord neurons, indicating that the photoaffinity labeling procedure does not cause gross cell damage. Neither was there any significant change in the mean gGABA (211M GABA), which was 2.6 ± 0.6 nS (n = 5) in untreated cultures, and 3.1 ± 0.6 nS (n = 10) in photoinactivated cultures, indicating that photoaffinity binding of FNZM to BZD receptors does not compromise the capacity of the GABA receptor to respond to GABA. When photoinactivated neurons were exposed to 300 11M CDPX, gGABA (5 ILM GABA) was increased only about 2-fold, compared to a 4 to 5 fold enhancement in untreated cultures (fig. 2). The change in potentiation of gGABA was not due to a direct effect of ultraviolet light nor to incomplete washout of FNZM, since potentiation was unaltered when cultures were exposed to FNZM without UV or UV-irradiated without FNZM. Thus, photoinactivation inhibits enhancement of gGABA by CDPX. Dose-response studies (fig. 3) revealed that the CDPX dose-response curve was shifted to the right, while the maximum enhancement "max was reduced, in qualitative agreement with the results of (3H]FNZM binding
176
Percent enhancement ( a)
Percent enhancement ( a)
llOO
•
131 (6)
(14)
(5)
400
300
200
(10)
[c DPXl, M
100
0 __
eonl'OI$
i~Ied
l.=llllllnl
&~~Ied ~~vFNZM
Fig. 2. Electrophysiology: photoinactivation inhibits enhancement of gGABA. Cultures were photoinactivated with FNZM. then assayed for enhancement of gGABA in the presence of 300 11M CDPX. Three different sets of control cultures were also tested: untreated. FNZM but no UV. and UV but no FNZM. The various controls did nOI dirrer significantly with respecl 10 enhancement of gGABA (solid control bar is pooled results from all 3 control procedures). Error bars indicate S.E.M.
studies. The increase in the CDPX ECso from 54 to 105 p.M (table 1) was apparently less striking than the over lO-fold increase in the K d for (3H]FNZM binding; however, it should be noted that the ECso and a max for enhancement of gGABA by CDPX were estimated on the basis of the assumption that enhancement of gGABA conforms to a hyperbolic dose-response relationship. While acceptable fits to the electrophysiological data were obtained by this method, the results of (3H]FNZM binding to photoinactivated membranes were clearly inconsistent with the hyperbolic model. Due to limited solubility of CDPX in recording medium we were unable to determine whether enhancement of gGABA by CDPX de-
Fig. 3. EleCtrophysiology: photoinactivation shifls CDPX dose-response curve to the right. Representalive examples of single-neuron dose·response curves are shown. Enhancement of gGABA (a) is plolted for two neurons. Open circles: control culture (exposed to FNZM but nOI UV); filled circles: photoinactivated culture. Dose-response curves were fitted to data by Eadie-Hofstee analysis. In Ihe experiment shown. the ECw was 48 IlM for the control neuron and 114 IlM for the photoinactivated neuron. while am.. values were respectively 180% and 342%.
viates from the hyperbolic model at high CDPX concentrations. Thus, it is possible that the estimated ECso underestimates the true magnitude of the change in CDPX potency. 3.3. fJHjBCCM binding: effect of photo;nactivation
The p-carbolines DMCM and BCCM displace (3H]FNZM in competitive binding experiments TABLE 1 Electrophysiology: effect of photoinactivalion upon enhancement of gGABA in spinal cord cultures. Spinal cord cultures were inactivated as described in Methods. then assayed for enhancement of gGABA. ECso and am.. were estimated for each tesl neuron by Eadie-Hofstee analysis. Values given are mean ± S.E.M. n
Control Photoinactivated
54±12 105± 10
430±98 185±19
11 8
177
~ . TABLE 2
TABLE 3
Binding: I)HIBCCM binding to control and photoinactivatcd 20-day membranes. Extensively washed control or photoin· activated 20-day brain membranes were incubated 60 min on ice with the indicated concentration of [)HIDCCM. then filtered, Nonspecific binding. determined in the presence of 10 I'M DCCM. has been subtracted. Protein was determined by Lowry. Values listed are the mean ± S.E.M. of 6 delermina· tions (n = 6) in 2 separate experiments. fmoljmg protein Control 0.1
1.0
4.0
47± 3 332± 9 719±24
Inactivated
Ratio·
37± 3 263± 30 567 ± 51
0,78 0.79 0.79
• Ina"tivated/control.
C"
and therefore may be assumed to bind either to the BZD site or to a closely coupled site. In contrast to BZDs such as FNZM or CDPX, however, BCCM and DMCM inhibit gGABA (table 3). Whereas reversible binding of subsaturating concentrations of (3 H)FNZM was typically reduced by at least 75% after photoinactivation (fig. 1), the binding of PH)BCCM was within 20% of control (table 2). Thus, BCCM binding is relatively insensitive to photoinactivation.
3.4. Elecrrophysiology: effecr of pho/OinacrivQrion on {J-carboline action
,
Given that (lH)BCCM binding is resistant to photoinactivation, would the same apply to inhibition of gGABA by BeCM? To investigate the effect of FNZM photoinactivation of BZD sites on modulation of gGABA by BCCM, cultures were photoinactivated and the ability of BCCM (1 14M) to inhibit gGABA was measured by pressure ejection of BCCM followed by GABA. Photoinactivation did not reduce the inhibitory effect of BCCM on gGABA (table 3A). Similar results were obtained with DMCM (table 3B). Since (3H]BCCM binding can be displaced by BZDs in both control and photoinactivated membranes, BZDs should reverse the inhibition of gGABA by {J-carbolines. This was in fact observed; the inhibitory effect of 30 nM DMCM on gGABA was abolished in the presence of 30 14M c1onazepam. The capacity of
Modulation of gGABA in control and photoinactivated spinal cord cultures, The indicated modulator (c1onazepam. DCCM. or DMCM) was applied by pressure ejection. followed by a pulse of GADA at the indicated concentration. Percent en· hancement/inhibition of gGADA (a) was calculated as described in Methods. Test condition
Control (a)
Photoinactivated (a)
(A) Inhibition of gGABA by DCCM J pM BCCM 10 pM GADA - 53 ± 9 (4) - 51 ± 5 (5) (8) Inhibition of gGABA by DMCM • .lOp,\.( DMCM 50 pM GA8A -45 ± 1(2) -42±23 (2) 500 pM GABA - 31 ± 5 (2) -32± 5 (2) }O pM DMCM + 30 pM clO1Ul:f!pUm 50pMGABA +57±31(2) -5±18(2) 500 pM GADA +6± 3 (2) -0.4± 2 (2) • For the experiments in group B. 30 pM picrotoxin was prescnt to reduce the magnitude of gGABA. This permitted unambiguous bridge balance and improved precision at elevated GADA concentrations. Although gGABA was reduced in the prescnce of picrotoxin. the value of II was nOI affecled (Chan. unpUblished results).
c10nazepam to antagonize inhibition of gGABA by DMCM was probably not due to the enhancemenl of gGABA by c10nazepam counterbalancing the inhibitory effect of DMCM, since antagonism between clonazepam and DMCM was observed at both 50 and 500 14M GABA, even though c10nazepam alone did not increase gGABA at the higher GABA concentration. Antagonism between DMCM and c10nazepam persisted in photoinactivated cultures, even though enhancement of gGABA by c10nazepam alone was inhibited by photoinactivation.
4. Discussion The interaction of a pharmacological agonist with its receptor to produce a physiological response requires a minimum of two steps: an initial binding event followed by a conformational change (or series of changes) in the receptor that ultimately leads to the observed effect. The observed dissociation constant, K d , determined from binding of a radiolabeled agonist reflects the net free
178
energy change for the overall reaction (Franklin. 1980; Jencks. 1983) and any change in the reaction sequence is likely to alter the apparent affinity. Thus. the reduced affinity for BZDs observed after photoinactivation could be due to a change in the initial binding event or to a change in subsequent steps leading to enhancement of gGABA or to a combination of both effects. To distinguish among these possibilities it is necessary to examine the effect of photoinactivation on the functional characteristics of the BZD receptor. If the effect of photoinactivation is limited to the BZD recognition site. then potentiation of gGABA should still occur, but greater BZD concentrations would be required to compensate for lower affinity binding. If, instead. reduced affinity for BZDs is associated with blockade of subsequent steps in the chain of events leading to enhancement of gGABA, then photoinactivation should eliminate enhancement of gGABA. Since the BZD receptor is coupled to the GABA receptor, an additional possibility is that photoinactivation might damage the GABA receptor, resulting in a diminution of the GABA response. It is at present unclear why the number of sites which can be irreversibly photoaffinity labeled with (3 HJFNZM is only about a quarter of the number of sites which can be reversibly labeled. nor is it known how the photoaffinity labeling procedure modifies the affinity of the remaining reversible binding sites. It has been suggested (Thomas and Tallman. 1983) that irreversible binding of FNZM to one BZD site in a 4-site complex could influence associated sites so as to reduce affinity for BZDs. This could occur if photoaffinity labeling induces an allosteric change in the conformation of neighboring sites or, conversely, if photoaffinity labeling blocks a conformational change normally associated with BZD binding. An alternate suggestion is that the BZD binding sites that do not irreversibly bind FNZM are directly modified in some other way in the course of the photolabeling reaction, and that this modification inhibits BZD binding (Herblin and Mechem, 1984). Scatchard analysis of (3H]FNZM binding to homogenates of 20-day embryonic chick brain suggests that binding sites are homogeneous in affin-
ity in control membranes. but heterogeneous in photoinactivated membranes. If the decrease in affinity for DZDs after photoinactivation is due to interactions between irreversibly labeled sites and the remaining reversible binding sites. this result argues that some reversible sites are more closely coupled than others to the sites which irreversibly bind FNZM. If. instead, reduced affinity reflects some other binding site modification distinct from irreversible labeling, then the results argue that the surviving BZD binding sites are not all modified in the same way. There was no significant change in gGABA after photoinactivation. indicating that photoinactivation did not compromise either the GABA recognition site or its coupling with chloride transport. Potentiation of gGABA by CDPX was reduced after photoinactivation, partly in consequence of a ca. 2-fold increase in the EC 50 for enhancement of gGABA. There was also a 43% decrease in the extrapolated maximum enhancement (am",,)' a somewhat larger change than the 25% decrease which would be expected if the only sites completely inactivated are those which become covalently linked to FNZM. It is possible that the decrease in am," is exaggerated by the assumption of a hyperbolic dose-response relationship. Scatchard analysis of (3HJFNZM binding to photoinactivated membranes suggests that BZD binding sites in photoinactivated membranes are heterogeneous in affinity. If this applies also to enhancement of gGABA by CDPX, then the am"" and EC50 estimated from Eadie-Hofstee analysis will underestimate the true values. and are best regarded as lower limits. The results thus support the conclusion that for most of the surviving BZD receptors photoinactivation affects only the initial binding of BZDs, leaving intact the coupling between BZD binding and enhancement of gGABA. This conclusion is in agreement with that of Brown and Martin (1984), who found that photoaffinity labeling does not eliminate enhancement of reversible diazepam binding by GADA. In contrast to the results obtained with (3 H]FNZM. binding of the tJ-carboline [3 H]BCCM was only slightly reduced after photoinactivation. Similar results have been reported for inactivation of BZD receptors in rat brain membrane homo-
"
179
..
genates by photoaffinity labeling (Gee and Yamamura, 1982; Hirsch, 1982; Karobath and Supavilai. 1982: Mohler, 1982: Thomas and Tallman, 1983) or exposure to irazepine. an alkylating BZO (Skolnick et a!., 1982). The resistance of the fJ-carboline binding site to treatments which inactivate BZO binding is somewhat paradoxical in view of the fact that fJ-carbolines and BZDs appear to compete for binding to membranes (Braestrup and Nielsen, 1981: O'Brien et aJ.. 1981: Sieghart et aJ.. 1983). It may be that BZO sites and fJ-carboline sites are physically separate, and the apparent competition for binding is the product of allosteric interactions; alternatively. fJ-carbolines and BZDs may bind to the same physical region of the receptor. but the structural determinants of fJ-carboline binding may differ from those that control BZO binding. In functional studies. BCCM proved to be an inhibitor of gGABA. If the effects of photoinactivation are limited to the initial binding step. inhibition of gGABA by BeCM should also be resistant to photoinactivation, in agreement with observation. Similar results were obtained with a second inhibitory fJ-carboline, DMCM. The inhibitory effects of DMCM could be counteracted by the potent benzodiazepine c1onazepam, indicating that photoinactivation does not abolish the mutual antagonism between BZOs and fJ-carbolines. In conclusion. the effects of photoinactivation on modulation of the GABA response are consistent with the hypothesis that the photoaffinity labeling reaction modifies BZD recognition sites. but leaves intact the mechanism by which binding of BZOs and fJ-carbolines is transduced to enhancement or inhibition of the GABA response.
References
"
Braestrup. C. and M. Nielsen. ]981. GABA reduces hinding or 3 H-methyl beta·carboline-3-carboxylate to brain benzodiazepine receptors. Nature 294. 472, Braestrup. C.. M. Nielsen and T. Honore. ]982a. Binding of r3 H\-DMCM. a convulsive benzodiazepine ligand. to rat brain membranes. Preliminary studies. J. Neurochem. 4]. 454. Braestrup. C.• M. Nielsen and C.E. Olsen. 1980. Urinary and brain beta-carboline-3-carboxylates as potent inhibitors of brain henzodiazepine receptors. Proc. Natl. Acad. Sci. U.S.A. 77. 2288.
Braestrup. c.. B. Schmiechen. G. Neef. M. Nielsen and E.N. Petersen. 1982b, Interaction of convulsive ligands with ben· zodiazepine receptors. Science 2]6. 1241. Brown. c.L. and I.L. Martin. ] 984. Photoaffinity labelling of the benzodiazepine receptor compromises the recognition site but not its effector mechanism. J. Neurochem. 43. 272. Chan. C.V .. T.T. Gibbs. L.A. Borden and D.H. Farb. ]983. Multiple embryonic benzodiazepine binding sites: evidence for functionality. Life Sci. 33. 2061. Choi. D.W.• D.H. Farb and G.D. Fischbach. 1977, Chlordiazepoxide selectively augments GABA action in spinal cord cell cultures. Nature 269. 342. Choi. D.W.• D.H. Farb and G.D. Fischbach. 198]. Chlordiazepoxide selectively potentiates GABA concuctance of spinal cord and sensory neurons in cell cultures. J. Neurophysiol. 45. 621. Farb. D.H.• O.K. Berg and G.D. Fischbach. ]979. Uptake and release of 13 H\gamma-aminobutyric acid by embryonic spinal cord neurons in dissociated cell culture. J. Cell BioI. 80. 651. Farb. D.H.• L.A. Borden. C.V. Chan. C.M. Czajkowski. T.T. Gibbs and G.D. Schiller. 1984. Modulation of neuronal function through benzodiazepine receptors: Biochemical and electrophysiological studies on neurons in primary monolayer cell culture. Proc. N.V. Acad. Sci. (in press). Franklin. T.J., 1980. Binding energy and the action of hormone receptors. Biochem. Pharmacol. 29, 853. Gee. K.W. and H.I. Vamamura. 1982. Differentiation of benzodiazepine agonislS and antagonists: sparing of [.IHlbenzodiazepine antagonist binding following the photolabelling of benzodiazepine receptors. European J. Pharmacol. 82. 239. Herblin. W.F. and c.c. Mechem. 1984. Short-wave ultraviolet irradiation increases photo-affinity labeling of benzodiazepine sites. Life Sci. 35.317. Hirsch. J.D.• 1982. Photolabeling of benzodiazepine receptors spares [)Hlpropyl beta-carboline binding. Pharmacol. Biochem. Behav. ]6. 245. Jencks. W.P.. 1983. On the economics of binding energies. Solvay Conference (in press). Jensen. L.F.. E.N. Petersen and C. Braestrup. ]983. Audiogenic seizures in DBA/2 mice discriminate sensitively between low efficacy benzodiazepine receptor agonists and inverse agonists. Life Sci. 33. 393. Karobath. M. and P. Supavilai. 1982. Distinction of benzodiazepine agonists from antagonists by photoaffinity labeling benzodiazepine receptors in vitro. Neurosci. Leu. 31,65. Macdonald. R.L. and J.L. Barker. 1978. Benzodiazepines specifically modulate GABA-mediated postsynaptic inhibilion in cultured mammalian neurons. Nature 271. 563. Mohler, H.• 1982. Benzodiazepine receptors: differenlial interllction of benzodiazepine agonisL~ and antagonists after photoaffinity labeling with nunitrazepam. European J. Pharmacol. 80. 435. Mohler. H.. M.K. Bauersby and J.G. Richards. 1980, Benzodiazepine receptor protein identified and visualized in brain tissue by u photoaffinity label, Proc. Nail. Acad. Sci. U.S.A. 71. 1666.
180 Oakley. N.R. and BJ. Jones, 1980. The proconvulsant and diazepam-reversing effects of ethyl-bela·carboline-3-carboxylate, European i Pharmacol. 68. 381. O'Brien. R.A., W. Schlosser, N.M. Spirt, S. Franco. W.O. Horst, P. Pole and E.P. Bonetti. 1981. Antagonism of benzodiazepine receptors by beta-carbolines. Life Sci. 29, 75. Schweri. M•• M. Cain, J. Cook, S. Paul and P. Skolnick. 1982, Blockade of 3-earbomethoxy-beta-carboline induced seizures by diazepam and the benzodiazepine antagonists Ro 15-1788 and CGS 8216. Pharmacol. Biochem. Dehav. 17, 457. Sieghart, W., A. Mayer and G. Drexler. 1983, Properties of (3Hlnunitrazepam binding to different benzodiazepine binding proteins. European J. Pharmacol. 88. 291.
Skolnick. P.. M. Schweri. E. KUller. E. Williams and S. Paul. 1982. Inhibition of [3H)diazepam and [lHlcarboethoxybeta-earboline binding by irazepine: evidence for multiple "domains" of the benzodiazepine receptor, J. Neurochem. 39. 1142. Thomas. J.W. and J.F. Tallman, 1983. Pbotoaffinity labeling of benzodiazepine receptors causes altered agonist·antagonist interactions. J. Neuroscience 3. 433. White, W.F.• M.A. Dichter and S.R. Snodgrass. 1981, Benzodiazepine binding and interactions with the GABA receptor complex in living cultures of rat cerebral cortex, Brain Res. 215. 162.
"
