and the Department of Biophysics, the Weizmann Institute ofscience, (Received 26 February 1973)

Transcription

1 J. Phy8iol. (1973), 234, pp With 10 text-ftgure Printed in Great Britain THE ROLE OF A REACTIVE DISULPHIDE BOND IN THE FUNCTION OF THE ACETYLCHOLINE RECEPTOR AT THE FROG NEUROMUSCULAR JUNCTION BY D. BEN-HAIM,* E. M. LANDAU AND I. SILMAN From the Department of Physiology and Pharmacology, Tel-Aviv University Sackler School of Medicine, Tel-Aviv, Israel, and the Department of Biophysics, the Weizmann Institute ofscience, Rehovot, Israel (Received 26 February 1973) SUMMARY 1. The effects of disulphide bond reduction and reoxidation on synaptic transmission in the frog neuromuscular preparation have been studied. 2. The amplitudes of end-plate potentials (e.p.p.s) and miniature e.p.p.s (m.e.p.p.s) were decreased irreversibly by the reducing agent dithiothreitol (DTT). Recovery of e.p.p.s and m.e.p.p.s could be achieved, however, by subsequent reoxidation employing 5,5'-dithio-bis-(2- nitrobenzoic acid) (DTNB). 3. No change in e.p.p. quantal content was produced by treatment with DTT and DTNB. The reduction of m.e.p.p. frequency found in DTT was attributed mainly to our inability to distinguish small m.e.p.p.s from the background noise. However, a genuine reduction in m.e.p.p.s frequency could not be ruled out. 4. The 'reactive' disulphide bond which is acted upon by DTT and DTNB could be located within a few Angstrom from the anionic site of the receptor for acetylcholine (ACh), by employing tre active-site directed reagent bromoacetylcholamine (BACA). 5. Reduction of the 'reactive' disulphide bond did not cause changes in the post-synaptic membrane input impedance nor was the e.p.p. reversal potential affected. Treatment with DTT and DTNB was found to modify only the conductance of the synaptic membrane. 6. No synaptic effects were produced by DTT in a non-cholinergic synapse, the crab neuromuscular preparation. 7. It is concluded that the receptor for ACh, besides including the well-known anionic site for binding quaternary ammonium groups, also * This work is part of a Ph.D. thesis by D. Ben-Haim, to be submitted to the Tel-Aviv University. It-2

2 306 D. BEN-HAIM, E. M. LANDAU AND I. SILMAN contains a unique 'reactive' disulphide bond. This bond controls synaptic conductance but not the relative permeability of the synaptic membrane to Na+ and K+. It is not yet clear whether the 'reactive' bond controls the interaction of ACh with the receptor or the translation of this interaction into ionic permeability.changes. INTRODUCTION There has been great interest recently in the characterization and isolation of the acetylcholine (ACh) receptor (Changeux, Kasai & Lee, 1970; Miledi, Molinoff & Potter, 1971; De Robertis, 1971; Eldefrawi, Eldefrawi & O'Brien, 1971; Fulpius, Klett, Cooper & Reich, 1972; Karlin, 1972). This receptor has been known for many years to comprise an anionic site, since a quaternary ammonium or a similar group is the essential feature of all cholinergic activating and blocking agents (Triggle, 1971, pp ). Recently, attempts have been made to define other reactive groups within the receptor complex (Changeux, Podleski & Wofsey, 1967; Kiefer, Lindstrom, Lennox & Singer, 1970; Waser, Hofmann & Hopff, 1970). One such group, a disulphide bond, was discovered by Karlin & Bartels (1966) in the eel electroplax preparation, where it was localized to within a few Angstrom from the anionic receptor site (Karlin & Winnik, 1968). When this bond (which might be called a 'reactive' disulphide bond) was reduced to free sulphydryl groups, the normal depolarizing effect of cholinergic agonists was blocked. This effect could only be reversed by oxidizing the free sulphydryl groups to reform the disulphide bond. However, the mechanism responsible for the disappearance of the effect of cholinergic agonists after disulphide bond reduction has remained obscure. This problem gains additional importance in view of the fact that a similar blockade by disulphide bond reduction has also been described in other cholinergic synapses: denervated rat muscle (Albuquerque, Sokoll, Sonesson & Thesleff, 1968), frog rectus (Mittag & Tormay, 1970), chick biventer (Rang & Ritter, 1971), frog sartorius (Del Castillo, Escobar & Gijon, 1971), leech muscle (Ross & Triggle, 1972). We therefore employed the frog neuromuscular preparation where the basic synaptic mechanisms are well understood (Katz, 1969). We first demonstrated that here also the 'reactive' disulphide bond had a similar localization to that found in the electroplax. Next, we could show that the 'reactive' disulphide bond did not control membrane permeability in general nor the relative permeability of the synaptic membrane to Na+ and K+. Rather, the synaptic-membrane conductance increase caused, by the transmitter was specifically decreased by the reduction of this bond. Finally, the 'reactive' disulphide bond appeared

3 DISULPHIDE BOND IN ACh RECEPTOR to be specific to cholinergic synapses since it could not be discovered in a non-cholinergic neuromuscular preparation. A preliminary note of our results has already been published (Ben-Haim & Landau, 1972). METHODS Preparationsq and 80lution8. When not stated otherwise, experiments were performed at room temperature, 20-24' C, on the sartorius nerve muscle preparation of the frog (Rana ridibunda) set in a 13 ml. Lucite tissue bath. Usually the bathing solution was frog Ringer of the following composition (maw): NaCl, 115; KCI, 2; CaCl2, 0.5; MgCl2, 2-5; Tris buffer (ph 8.0), 1. The high ph value was chosen to increase the rate of reduction by the sulphydryl reagent (Cecil, 1963, p. 394). Deviations from this standard Ringer will be indicated where appropriate in the Results section. Some experiments were performed on the rat phrenic nerve diaphragm preparation at room temperature employing a suitable Ringer solution (mm): NaCl, 137; KCl, 5; MgCI2, 5; CaCl2, 0-5; NaH2P04, 1; NaHCO3, 24; glucose, 1 1; Tris buffer (ph 8.0), 1. The preparation was set in a 4 ml. perfusion chamber (Hubbard, Jones & Landau, 1958) allowing continuous perfusion of Ringer equilibrated with 95% 02 and 5 % CO2. Experiments were performed also on a crab nerve muscle preparation (Ocipoda cursor, closer muscle of the leg) (Parnas & Samne, 1972). Here the composition of the Ringer was (mm): NaCl, 453; CaCl2, 34; MgCl2, 4'9; KC1, 12-8; Tris buffer (ph 8-0), 1. The perfusion chamber had a volume of 5 ml. Drugs. In this study we employed dithiothreitol (DTT, Sigma), 5,5' dithiobis-(2-nitrobenzoic acid) (DTNB, Sigma), hexamethonium (Fluka), gamma aminobutyric acid (GABA, Sigma), bromoacetylcholamine bromide (BACA) and bromoacetamide (BAA). The two latter materials were synthesized by one of us (I. S.). All drugs were dissolved in Ringer and applied by perfuming the preparation with ml. of the drug-containing Ringer. Stimulation and recording. The nerve supply to the preparation was stimulated supramaximally using a Grass 5 4 stimulator at a rate of 0-5 sec-1 (frog), 1 sec-1 (rat) or 10 sec-1 (crab). Miniature end-plate potentials (m.e.p.p.s.) and end-plate potentials (e.p.p.s.) were recorded intracellularly with 2-5 M-K citrate filled glass microelectrodes. Signals were displayed employing a preamplifier (NF1, Bioelectric) and a 565 Tektronix oscilloscope (Tektronix amplifier 3A9; the frequency range was usually set to d.c. to 10 khz). Data were stored on a magnetic tape (Hewlett Packard 3902) and permanently recorded on cine ifilm or on chart paper (Grass 79 polygraph) after eightfold reduction of the tape speed. Also, e.p.p.s were automatically averaged by a computer for averaging transients (TMC-CAT 400C) and then plotted with a X-Y recorder (Hewlett Packard 7035 B). In a number of experiments in the frog and in the crab two micro-electrodes were introduced under visual inspection into the same cell, 2-3 fibre diameters apart, one being used for recording and the other for passing current. In this way we could measure the effective membrane input impedance (Katz & Thesleff, 1957) and in the frog, also the e.p.p. reversal potential. In the latter experiment we used detubulated preparations, prepared according to Eisenberg, Howell & Vaughan (1971). Following their method, the Ca2+ and Mg2+ concentrations were both 5 mm. Determination of e.p.p. quantcsl content. This was done by counting the number of failures of synaptic transmission in 200 consecutive nerve stimulations. The calculation was performed according to Martin (1966). Determination of m.e.p.p. frequency. In a few experiments we were confronted with the necessity of counting m.e.p.p.s with reduced amplitudes. In order to

4 308 D. BEN-HAIM, E. M. LANDAU AND I. SILMAN minimize the loss of the smaller m.e.p.p.s in the background noise, we inserted a second micro-electrode into the cell under visual inspection and employed it to hyperpolarize the muscle cell membrane. RESULTS Modification of the end-plate potential by disulphide bond reduction The basic phenomenon that we studied is illustrated in Fig. 1. Here the effects of DTT (a disulphide bond reducing agent (Cleland, 1964), and DTNB), an oxidizing agent, were examined. First, we recorded and automatically averaged 110 e.p.p.s (Fig. 1A). The average control amplitude was usually very stable and could repeatedly be obtained with only minor variations. The resting potential of the cell was -85 mv. When DTT at a concentration of 1 mm was applied AI\ B C D Fig. 1. The effect of disulphide bond reduction and reoxidation on e.p.p. amplitude. Each trace represents 110 automatically averaged e.p.p.s. A, control; B, after 15 min in DTT (1 mm). 0, 15 min after wash out of DTT. D, lower trace, after 90 min in DTNB (1 mm); upper trace, after 120 min in DTNB. Resting potential -85 mv. Calibration, voltage 2-5 mv; time 5 msec. to the preparation, a gradual reduction of the average e.p.p. could be observed (Fig. 1 B, at 15 min after application of DTT). At the same time the resting potential of the cell was not altered. When DTT was washed out from the preparation (Fig. 1C) no increase in the e.p.p. amplitude could be observed. On the contrary, the effect of the DTT had increased during the wash-out period (lasting about 15 min) and the e.p.p. amplitude was even further reduced. When the oxidizing agent DTNB (1 mm) was added to the preparation, the e.p.p. amplitude promptly started to increase and returned to almost 90 % of the control value within 90 min, and to 95 % within 120 min (Fig. I D). The resting potential of the cell remained -85 mv throughout the experiment. Control experiments with DTNB alone showed no significant effect of this agent on the e.p.p. amplitude. The experiment of Fig. 1 was typical of all the experiments where DTT and DTNB were applied. The average reduction of the e.p.p.

5 DISULPHIDE BOND IN ACh RECEPTOR 309 size by 1 mm-dtt in six experiments was to % (mean + 1 S.D.) of the control. In these experiments the restitution of the e.p.p. amplitude by 1 mm-dtnb acting for half an hour or more (usually an hour) was on the average to % (mean + 1 S.D.) of the control (i.e. pre-dtt) value. The restitution was probably even better than that, because after long periods of impalement a small deterioration of the resting potential often occurred and this would reduce the amplitude of the observed e.p.p.s. After restitution with DTNB a second cycle of reduction and oxidation could readily be obtained. Two similar experiments to that shown in Fig. 1 were performed in the rat phrenic nerve diaphragm preparation with similar results. Thus, we could not support the finding of Mittag & Tormay (1970) that DTT did not affect the cholinergic receptors at the rat neuromuscular junction. This discrepancy may have been due to a difference in experimental procedure. Mittag & Tormay recorded muscle contractions whereas we directly recorded the synaptic potentials, a more sensitive index of synaptic activity. The effects of disulphide bond reduction on spontaneous transmitter release In order to determine whether the effects shown in Fig. 1 had a pre- or post-synaptic origin we first examined the effects of DTT and DTNB on miniature end-plate potential (m.e.p.p.) amplitudes. When 01 mm-dtt was applied for 90 min, the amplitude of the m.e.p.p.s decreased by more than half (Fig. 2A, B). This effect could be reversed by the application of 0.1 mm-dtnb (Fig. 20). M.e.p.p. amplitudes were measured before and after DTT (0.1 mm, min) in seven experiments and the average m.e.p.p. amplitude in DTT was % (mean + 1 S.D.) of control. In two other experiments the m.e.p.p. amplitudes after DTT (0.5-1 mm, 8-15 min) were too small to be measured. In all experiments DTNB, at concentrations equal to those of DTT, could produce a recovery of the m.e.p.p. amplitudes within min. The reduction of m.e.p.p. amplitudes by even small concentrations of DTT indicated that this reagent had a post-synaptic effect (see del Castillo & Katz, 1956). Drugs which block ACh synthesis can cause a reduction of the m.e.p.p. amplitude, provided that the rate of transmitter release is strongly accelerated (Elmqvist & Quastel, 1965). However, in the experiments just described the nerve was not stimulated and the frequency of m.e.p.p.s was small ( < 15 sec-1 and usually < 4 sec-'). When looking at the results of experiments such as in Fig. 2, we had the impression that not only the m.e.p.p. amplitude but also m.e.p.p. frequency became reduced by DTT. This would have been interesting in view of the findings of Werman, Carlen, Kushnir & Kosower (1971) showing presynaptic effects of a sulphydryl oxidizing agent. M.e.p.p. frequency was measured before and after treatment with DTT (0 1-1 mm,

6 310 D. BEN-HAIM, E. M. LANDAU AND I. SILMAN min) and was found to decrease to 62+28% (mean+1 S.D.; n = 10) of control. In four of these experiments hyperpolarizing currents were passed from a second intracellular micro-electrode (see Methods) to increase m.e.p.p. amplitude. We constructed m.e.p.p. amplitude histograms, before and after treatment with DTT. In all cases except one, the distribution of m.e.p.p.s after DTT became very skewed, indicating that some m.e.p.p.s may have been lost in the background noise. The apparent decrease in m.e.p.p. frequency may thus have been post-synaptic in origin, although a presynaptic effect remains an intriguing possibility. A.1 B C Fig. 2. The effect of disulphide bond reduction and reoxidation on m.e.p.p. amplitude. A, control; B, 90 min in 0 1 mm-dtt; C, 90 min in 0 1 mm- DTNB. 1X6 mm-ca2+, 25 mm-mg2+. Resting potential -95 mv. Calibration, voltage 0 5 mv; time 50 msec. DTNB in concentrations equal to the DTT caused a recovery of m.e.p.p. frequency, presumably due to its post-synaptic effects. However, in three experiments where DTNB alone at concentrations between mm was applied for 120 min or more, m.e.p.p. frequency increased fold and bursts of m.e.p.p.s were observed. This effect of DTNB, an oxidizing agent which probably penetrates poorly into cells, could be similar to the effect of diamide described by Werman et al. (1971). Effect on the quantal content of e.p.p.s In order to determine whether the effects of DTT and DTNB on the e.p.p. amplitude had a purely post-synaptic origin or whether some

7 DISULPHIDE BOND IN ACh RECEPTOR 311 presynaptic change was involved as well, we examined the effects of these agents on the e.p.p. quantal content. In a typical experiment (Fig. 3) the quantal content (m) was determined by the failure method (see Methods) in preparations paralysed by excess Mg2+ and reduced Ca2+. After determining the quantal content of the e.p.p.s, DTT (1 mm) was added by slow perfusion (Fig. 3, first horizontal bar). After this treatment the e.p.p. size decreased to 40% of the control value (Fig. 3, first and second insets on the left). At the same time no m 4 2 I II I N_ Time (mi.n) Fig. 3. The effect of disulphide bond reduction and reoxidation on e.p.p. quantal content. 0 5 mm-ca2+, 10 mm-mg2+. Ordinate: quantal content (in). Abscissa: time (min). Horizontal bars: periods of perfusion; 1st, 1 mm-dtt; 2nd, 1 mu-dtnb. Dashed vertical lines denote the end of the application of DTT and DTNB respectively. Vertical bars: ± 2 S.D. Insets: superposition of 20 e.p.p.s in each. From left to right: control, end of perfusion with DTT, 30 min in DTNB, t00 min in DTNB. Resting potential -90 to -80 mv. Calibration, height of inset 1-5 mv, width of inset 15 msec. reduction of 'm' could be found. When DTNB was added to the preparation (the period of slow perfusion is indicated by the second horizontal bar in Fig. 3) the e.p.p. amplitude returned to 87 % of control within 1 hr (third and four insets of Fig. 3). The value of 'm' showed some increase (40 %), although this was not significant. Similar results were found in seven more experiments. In all of them, there was a similar increase of

8 312 D. BEN-HAIM, E. M. LANDAU AND I. SILMAN 'in' (31 ± 12%, mean+ 1 S.D.) after prolonged exposure to DTNB. It was difficult to ascribe significance to this finding, because it may have been produced by a slow diffusion of Ca2+ out of the muscle fibre (Meiri & Rahamimoff, 197 1). The lack of effect of the two chemical agents on '' compared with their marked influence on the average e.p.p. size (Fig. 1), further supported the contention that their effect was primarily to modify the post-synaptic membrane. It therefore seemed reasonable to use the amplitude of the e.p.p. as a measure of post-synaptic changes produced by treatment with DTT and DTNB. Localization of the 'reactive' di-suiphide bond In the previous section we have shown that DTT decreases the e.p.p. amplitude by a post-synaptic mechanism. Since DTT is a specific sulphydryl reagent (Cleland, 1964) we can assume that many disulphide bonds in the membrane are affected by it. The questions arise whether the synaptic effects of DTT are mediated by the reduction of one specific disulphide bond, and whether this bond is located in the vicinity of the anionic site of the ACh receptor. To answer these questions we employed the active-site directed reagent bromoacetylcholamine bromide (BACA- BrCH2CONH(CH2)2N+(CH3),Br-; Kalderon & Silman, 1971). This chemical combines the properties of an alkylating agent and a cholinergic antagonist. By virtue of its quaternary ammonium moiety it can complex specifically and reversibly with the anionic site of the receptor for ACh, and owing to its bromoacetyl. moiety it can react irreversibly with some reactive group (probably a sulphydryl, cf. Silman & Karlin, 1969) near the anionic site to form a covalent bond. The formation of the initial reversible complex will increase the concentration of the labelling reagent in the vicinity of the anionic site as compared to its concentration in the free solution. Therefore, if sulphydryl groups are produced by the action of DTT very close to the anionic site, we expect BACA to react with them and thus prevent their reoxidation by DTNB. The effect of BACA should be prevented if the ACh receptor is protected by the presence of an excess of a reversible cholinergic agent, e.g. hexamethonium. Moreover, BACA should be a much more potent alkylating agent than a similar compound without the quaternary ammonium moeity (bromoacetamide, BAA). We first examined the effect of BACA alone in concentrations up to 3 x 10-5M acting for 30 min. It had a small blocking effect, which was completely reversed by washing out the reagent. We then examined the effect of BACA after DTT (Fig. 4). In this experiment we recorded an average control e.p.p. (Fig. 4A) and then applied 2 mm-dtt for 20 min and the average e.p.p. amplitude became smaller (Fig. 4 B, upper trace). We then added 6 x IO-' m-baca, which

9 DISULPHIDE BOND IN ACh RECEPTOR31313 further reduced the e.p.p. size (Fig. 4 B, lower trace). After 20 mmin BACA, 4 mm-dtnb were added and the synapse further observed for 70 min (Fig. 40). At this time, the e.p.p. amplitude had recovered to only 48% of control (Fig. 40, lower trace) whereas in three matching control experiments (cf. also Fig. 1) DTNB acting after DTT alone produced an average recovery of e.p.p. size to 92 % of control (range %). The failure of DTNB to produce almost complete recovery of the e.p.p. amplitude after BACA was found in five experiments, the average recovery after DTNB being 39±+ 9% (mean ± 1 S.D.) of control. We therefore concluded that BACA partly prevented the restitution of the e.p.p. size normally produced by DTNB. A B C / Fig. 4. The effect of BACA on disulphide bond reduction and reoxidation. Each trace represents 100 automatically averaged e.p.p.s. A, control. B, top trace, after 20 mins in DTT (2 mm) and washout; bottom trace, 20 mini in BACA (6 x 10-7M). C, top trace, 20 min in DTNB (4 mm); bottom trace, 70 min in DTNB. Resting potential: - 76 to - 74 mv. Calibration, voltage 1 mv, time 5 msec. A test of the specificity of BACA as an active-site directed reagent could be the ability of a reversible cholinergic blocking agent to protect the ACh receptor from the action of BACA. Protection of the ACh receptor from BACA by hexamethonium was found in four experiments, the results of one being shown in Fig. 5. After recording control e.p.p.s (Fig. 5A), 2 mm-dtt was applied for 20 min. The average e.p.p. amplitude was seen to decrease gradually (Fig. 5 B, upper trace). Then 6 x 10-7 M-BACA together with 1PS mmi hexamethonium. were added for 20 mini. The addition of both drugs promptly caused a disappearance of the e.p.p. (Fig. 5 B, lower trace). When the drugs were washed out and 4 mm-dtnb added for 70 min the average e.p.p. size was found to increase gradually to 80% of the control value (Fig. 50C). The resting potential was - 70 to - 72 mv throughout the experiment. The average recovery of the e.p.p. size after 70 min in DTNB was found in three experiments to be 85% of control (range 76 to 100%). In a fourth experiment the cell was lost after

10 314 D. BEN-HAIM, E. M. LANDAU AND I. SILMAN 45 minl in DTNB when the e.p.p. amplitude had already recovered to 60% of control. The effect of DTNB was thus similar to that found in the absence of BACA. We therefore concluded that the receptor could be protected from the action of BACA by hexamethonium. However, when concentrations of BACA higher than 10--6m were applied after treatment with DTT, the recovery of the e.p.p. amplitude caused by DTNB was much less than 39% and hexamethonium in concentrations up to 1-5 mmw offered no effective protection of the receptor. This could be explained if A c Fig. 5. Prevention of the BACA effect by hexamethonium. Same as Fig. 4 but hexamethonium (1.5 mm) was applied together with BACA (bottom trace of B). Resting potential, - 72 to - 70 mv. the affinity of BACA to the reduced receptor were much larger than that of hexamethonium. A greater range of hexamethonium concentrations and perhaps other cholinergic agents would have to be examined to elucidate this point. Next we compared the effect of BACA to that of a similar alkylating agent with no quaternary ammonium moiety, namely BAA. In this experiment (Fig. 6) the average e.p.p. size was reduced by DTT (1 mm, 25 min) (Fig. 6A, B). Then 1 mm-baa was added for 30 min (Fig. 60) and finally DTNB (1 mmx, 40 min) was introduced (Fig. 6D). The restitution of the e.p.p. amplitude by DTNB was to 84%, a value not different from that found on the average without BAA. The average recovery in three experiments with BAA was to 91 % of control (range %). We concluded that BAA, at a concentration many times greater than BACA, did not produce any effects on the ACh receptor. Therefore, the alkylating effect of BACA seemed to have been potentiated very markedly by its quaternary ammonium moiety. The results of the above experiments (Figs. 4-6) show that it is possible to specifically label the sulphydryl groups produced by DTT. This fact indicates that these sulphydryl groups are located at a molecular distance

11 DISULPHIDE BOND IN ACh RECEPTOR 315 away from the anionic site of the ACh receptor. In this respect the frog neuromuscular junction resembled the electroplax preparation (Kalderon & Silman, 1971). A ~ikx B C D Fig. 6. The effect of BAA on disulphide bond reduction and reoxidation. Each trace represents 110 automatically averaged e.p.p.s. A, top and bottom traces: two controls, the interval between them 20 min. B, top and bottom traces: 10 and 20 min in DTT (1 mm) respectively. a, 30 min in BAA (1 mm). D, bottom and top traces 10 and 40 min in DTNB (1 mm) respectively. Resting potential: -75 mv. Calibration: voltage 1 mv; time 5 msec. Effects of methonium compounds after DTT treatment One of the striking findings of Karlin (1969) on the electroplax was that after treatment with DTT, the reversible cholinergic antagonist hexamethonium acted as an agonist, and the reversible cholinergic agonist decamethonium became a more efficient agonist. This indicated that DTT treatment did not simply inactivate the ACh receptor, but rather changed its structure in a specific manner. We did six experiments with hexamethonium (3 x 10-5 M and 1 mm) and with decamethonium (1 mm) after DTT, but no change in the resting membrane potential could be observed.

12 316 D. BEN-HAIM, E. M. LANDAU AND I. SILMAN The resistance of the muscle fibre membrane A possible, if unlikely, explanation of the action of DTT was that it might reduce the response to ACh by changing the resistance of the fibre membrane. This was tested by examining the effects of DTT and DTNB on the input resistance, as measured by the methods described by Katz & Thesleff (1957). In seventeen such experiments, the input resistance was foundto be (mean + 1 S.D., n = 17) MQ. When DTT ( mm) had been applied from 15 to 100 min, the input resistance was (mean + 1 S.D., n = 17) MO. The input resistance in DTNB ( mm, acting for min) was (mean + 1 S.D., n = 8) MO. The reversal potential of the e.p.p. A second possible role for the reactive disulphide bond could be to control the permeability of the ionic channel through which the synaptic current flows. One experimental approach to this problem is to determine the transmitter reversal potential which is a measure of the relative permeability of the synaptic membrane to Na+ and K+ (Takeuchi & Takeuchi, 1960). Thus, if the reduction of the 'reactive' disulphide bond caused the reversal potential to be shifted to a more negative level, say from - 15 to -40 mv, this could perhaps account for the observed diminution of the e.p.p. amplitude. We tested this possibility by inserting two micro-electrodes into the same cell, employing one to record e.p.p.s and the other to pass polarizing current. The muscle fibres were detubulated (see Methods) and therefore had rather low resting potentials (-50 to -32 mv in nine experiments). However, the input impedance of the fibres was in the normal range. A typical experiment is shown in Fig. 7. In Fig. 7A (top trace) we see the normal e.p.p. and in Fig. 7B (top trace) we see the reduced e.p.p. after the addition of 1 mm-dtt for 25 min. The resting potential of the cell was -37 to -32 mv. Each of the bottom traces in Fig. 7A shows an e.p.p. obtained at a different membrane potential level. As the muscle membrane became more depolarized, the e.p.p.s became smaller, and when the membrane potential was equal to the reversal potential (-5 mv), no e.p.p. was seen. When the membrane potential was more positive (inside vs. outside) than -5 mv, the sign of the e.p.p. was inverted (cf. Takeuchi & Takeuchi, 1959, 1960). Reversal potentials were often more positive than -15 mv (average in nine experiments: -10 mv, range -21 to + 6 mv). This, as well as the low resting potentials, may be explained by the cells losing K+ during detubulation (Gage & McBurney, 1972). In the presence of DTT (Fig. 7 B) although the e.p.p.s were smaller, the reversal potential was not significantly altered. The fact that the reversal potential was unchanged may

13 DISULPHIDE BOND IN ACh RECEPTOR 317 serve as an indication that DTT did not affect the relative permeability of the ionic channel responsible for the end-plate current (see also Discussion). The effect of DTNB on a DTT-treated cell can be seen in Fig. 7C, D (this is a different cell from that of Fig. 7A, B). Here, 1 mm-dtnb. acting for 30 min, produced an increase in the e.p.p., again without affecting the reversal potential. Similar results with DTT were obtained in seven experiments. In only one was there a change in the reversal potential as compared to control, from -24 to -15 mv. Also, no change A B C D Fig. 7. The effects of disulphide bond reduction on the reversal potential of the e.p.p. Top traces in A, B. a, D: e.p.p.s at the normal testing potential (At - 37 mv; B m.v; a, - 38 mv; D, - 36 mv). Other traces in A, B. a, D: e.p.p.s at different resting potential levels, the upper trace in each series is at the most depolarized level. A, control, reversal potential -5mV. B. 25minDTT (I1 mm); reversal potential -5 mv. C, 25 min in DTT; reversal potential -4 mv. D, 30min in DTNB (I mm); reversal potential -4 mv. A and B in the same cell; a and D in another cell. Calibration: voltage IO mv, time 5 msec. was found after DTNB in three experiments. In a fourth experiment the reversal potential changed from -15 to -9 mv. These occasional shifts in the reversal potential were either in the wrong direction or too small to account for the effects of DTT and D~TNB, and can probably be disregarded. When the e.p.p. amplitudes from an experiment similar to that in Fig. 7 were plotted vs. the membrane potential level (Fig. 8a), a straight-line relationship was obtained, as was to be expected, over the range of membrane potentials examined (Takeuchi & Takeuchi, 1959;

14 i - l w s 318 D. BEN-HAIM, E. M. LANDAU AND I. SILMAN Kordav, 1969; Maeno, Edwards & Hashimura, 1971; Magleby & Stevens, 1972). When 1 mm-dtt was applied for 25 min no change in the reversal potential (-8 mv) was observed, the relationship remained linear, but its slope was decreased to 59% of control (Fig. 8b). The application of 1 mm-dtnb to the same cell for 15 min reversed the effect of DTT on the slope, again without changing the e.p.p. reversal potential (Fig. 8C). The slope in Fig. 8 was proportional to the synaptic conductance provided the e.p.p.s were corrected for non-linear summation (Martin, 1966; Katz & Miledi, 1972). When this correction was introduced, the reduction of the synaptic conductance by DTT was to 43-5% of control. The major effect of DTT would thus appear to be to reduce the over-all synaptic conductance change without affecting the relative permeability of the synaptic membrane to Na+ and K+. 12 a b \M C 8 \u 4 E W_\ _ *_ 0 Q1i -4-8 I~ I I I _ RP (mv) Fig. 8. The effects of disulphide bond reduction and reoxidation on the synaptic conductance from an experiment similar to that of Fig. 7. Ordinate: e.p.p. amplitude in mv. Abscissa: resting potential in mv. The resting potential (RP) which corresponds to the intersection point of the plot and the interrupted line is the reversal potential (-8 mv in A, B, C). a, Control; b, 25 min DTT (1 mm); c, 15 min DTNB (1 mm). Resting potential of cell, -46 to -44 mv. The effect of DTT on non-cholinergic synapses An interesting problem was whether the 'reactive' disulphide bond described in the previous sections was specific to the cholinergic receptor or whether it could be found in other receptors as well. In order to answer

15 DISULPHIDE BOND IN ACh RECEPTOR 319 this question we turned to a crab neuromuscular preparation where both excitatory and inhibitory synapses can be found (Atwood, 1967; Parnas & Sarne, 1972). The inhibitory transmitter here is gamma aminobutyric acid (GABA) and the excitatory transmitter is perhaps glutamate (Takeuchi & Takeuchi, 1964, 1966a, b). We first examined the effects of GABA on the membrane input impedance of the crab muscle cells. In this preparation the effect of GABA desensitizes very rapidly (Epstein & Grundfest, 1970; Parnas & Sarne, 1972) and we had to perform the measurements within a minute after the exchange of the control solution to a solution containing GABA. The effect of a current pulse (Fig. 9, bottom traces) on the membrane potential can be seen both before (Fig. 9A) and after (Fig. 9a) the addition of GABA (3.5 x 10-5 g/ml.). The effective membrane impedance fell to 78 % of control. After this, A a B g b Fig. 9. Effects of disulphide bond reduction on the post-synaptic response to GABA at the crab neuromuscular junction: Lower traces: the input current pulse (2 x 10-6 A). A, a: control changes in resting potential before and after GABA (5 x 10-5 g/ml.) respectively. B, b: same as A, a, but 60 min after DTT (1 mm). Calibration: voltage 0.5 mv, time 25 msec. GABA was washed out and DTT at a concentration of 1 mm was applied for 60 min. It can be seen (Fig. 8 B) that the membrane impedance was now larger, the increase being at least 80 % of control. This value was an underestimate because the voltage trace in Fig. 9 B had not reached its full height. A concomitant increase in the membrane time constant is also evident. The effect of GABA, in the same concentration as before, was now slightly increased (Fig. 9b), the reduction of the input impedance

16 320 D. BEN-HAIM, E. M. LANDAU AND 1. SILMAN being to 56% of control. An increase in membrane impedance in DTT was found in six out of eight experiments, where the effects of the reducing agent (1-2 mm for min) were examined. In two other experiments no effect was found. The average increase was % (mean + 1 S.D., n = 6) of control. The effect of DTNB (1 mm for min) after DTT was tested in five experiments. In four of these, membrane impedance became reduced by % (mean + 1 S.D., n = 4). In the fifth experiment no effect was found. The effect of GABA (10-5 to 5 x 10-5 g/ml.) after treatment with DTT was examined in three experiments. In all of A B I,'..j*.**i :4 ri t- i - 8.\ I- L... I.-IF- I t - Fig. 10. Effects of disulphide bond reduction on the presynaptic response to GABA at the crab neuromuscular junction. Each trade represents 200 automatically averaged j.p.s. A, control; B, after 65 min in DTT (2 mm). Top traces, before GABA; middle traces, 3 min after GABA (2 x 10-5 g/ml.). Bottom traces, after wash out of GABA. Calibration: voltage 0.5 mv, time 5 msec. them the effect of GABA was slightly enhanced. We could conclude that DTT treatment did not block the post-synaptic response to GABA and that the slight increase in the response was due to a non-specific increase of the membrane impedance. We could also test the effects of DTT on synaptic transmission in this preparation. To this end the nerve was stimulated at 10 c/s and 200 junctional potentials (j.p.s.) were averaged (Fig. 10A, upper trace). When GABA (2X10-5 g/ml.) was added to the bathing solution a reduction of the j.p. amplitude occurred (Fig. 1OA middle trace) which did not show signs

17 DISULPHIDE BOND IN ACh RECEPTOR 321 of desensitization. The origin of this effect of GABA is probably presynaptic (Epstein & Grundfest, 1970; Parnas & Sarne, 1972). Washing the GABA away resulted in a prompt recovery of the j.p. amplitude (Fig. 10A, bottom trace). This sequence was repeated in the same cell after treatment with DTT (2 mm for 65 min, Fig. lob). It can be seen that the j.p. amplitude was slightly increased (114-fold), but GABA had the same relative effect as before. In ten out of eleven experiments treatment with DTT (1-2 mm for min) increased the j.p. by % (mean + 1 S.D., n = 10), whereas in the eleventh experiment no effect was found. This increase was of the same magnitude as the increase in the fibre input impedance (55 %), and it thus seemed likely that the increase in the j.p. was a consequence of the increased fibre input impedance. The presynaptic effect of GABA after treatment with DTT (Fig. 1OB) was found to persist in three experiments. We conclude that DTT probably had no effect on the specific receptors for GABA and glutamate in this preparation. DISCUSSION In the present study we have shown that the activity of the endogenous ACh released from the presynaptic membrane requires an intact disulphide bond in the post-synaptic membrane (Figs. 1, 2). This agrees with the conclusion of other workers drawn from experiments in the electroplax and other cholinergic preparations (see Introduction), but differs from the model put forward by del Castillo et al. (1971). These workers used the iontophoretic method of drug application, and the concentrations attained near the receptors are likely to have been greater than in the present experiments. Different chemical groups may thus have been affected (del Castillo, Bartels & Sobrino, 1972). The endogenous ACh reacts exclusively with junctional receptors which are reportedly different from the extrajunctional ones (Feltz & Mallart, 1971). It has already been demonstrated that the extrajunctional receptors encountered in denervated muscle are blocked by DTT (Albuquerque et al. 1968). The present results thus supplement the findings of Albuquerque et al. and demonstrate a similarity between the structure of both junctional and extrajunctional receptors. Further, our findings provide evidence that the disulphide bond mentioned above is located a few Angstroms away from the anionic receptor site for ACh. This can be concluded because BACA prevents the reoxidation of the receptor by DTNB (Fig. 4). The effect of BACA is specific and cannot be imitated by BAA, which lacks the quaternary ammonium moiety (Fig. 6). Also, the effect of low concentrations of BACA is prevented by hexamethonium which presumably protects the receptor

18 322 D. BEN-HAIM, E. M. LANDAU AND I. SILMAN by occupying the anionic site (Fig. 5). The structure of the ACh receptor in the frog myoneural junction is thus similar to the receptor in the electroplax (Kalderon & Silman, 1971). However, a difference between frog myoneural and electroplax receptors is indicated by our failure to detect a depolarizing action of hexamethonium after DTT treatment. Such an effect was found in the electroplax (Karlin, 1969) and in the chick biventer muscle (Rang & Ritter, 1971). However, the reduced cholinergic receptor in leech muscle was not affected by hexamethonium (Ross & Triggle, 1972). It thus appears that whereas a broad structural similarity exists between various nicotinic receptors, a certain amount of species difference can be discovered. We could find no evidence for a similar 'reactive' disulphide bond in the crab neuromuscular preparation, where the transmitters are GABA and probably glutamate (Takeuchi & Takeuchi, 1964, 1966a, b) (Figs. 9, 10). If the GABA or glutamate receptors are proteins, it is very likely that they also contain disulphide bonds. However, these bonds may either not be accessible to our reagents or not involved in the interaction between transmitter and receptor. A 'reactive' disulphide bond may thus be a specific feature of the nicotinic receptor for ACh although other receptors, including adrenergic as well as muscarinic, have yet to be examined. Our results indicate that the 'reactive' disulphide bond is probably not involved with the function of the synaptic ionic channels. This is shown by the failure of DTT and DTNB to affect the e.p.p. reversal potential (Figs. 7, 8). However, this result cannot exclude a concomitant reduction of both Na and K permeabilities of the synaptic membrane (see Werman, 1965). That the nature of the ionic channels is unaltered is also suggested by the observation that the relationship between the e.p.p. size and themembrane potential remains linear in DTT (Fig. 8) and no illinearities like those produced by procaine or by extreme changes in the membrane potential (Kordas, 1970; Maeno et al. 1971) are found. We thus see that only the synaptic conductance change produced by a given amount of ACh is decreased by treatment with DTT (Fig. 8). This decrease can be explained either by a reduced binding of ACh to the receptor (reduced 'affinity') or by a reduced permeability change caused by the AChreceptor interaction (reduced 'efficacy'). It may be worth while to employ the statistical method of analysing ACh potentials (Katz & Miledi, 1972) to differentiate between these two possibilities.

19 DISULPHIDE BOND IN ACh RECEPTOR 323 We wish to thank Professor S. Gitter for his constant help and encouragement. We are also grateful to Professor I. Parnas and Dr Y. Same for introducing us to the crab neurmuscular preparation. A grant to one of us (I.S.) from the Volkswagen Foundation is gratefully acknowledged. REFERENCES ALBUQUERQUE, E. X., SOKOLL, M. D., SoNEssoN, B. & THESLEFF, S. (1968). Studies on the nature of the cholinergic receptor. Eur. J. Pharmac. 4, ATWOOD, H. L. (1967). Crustacean neuromuscular mechanisms. Am. Zool. 7, BEN-HAIM, D. & LANDAU, E. M. (1972). Chemical modification of the receptor for acetylcholine in the frog neuromuscular synapse. Israel J. med. Sci. 6, 580. CECIL,, R. (1963). The role of sulfur in proteins. In The Proteins, vol. 1, ed. NEURATH, H. New York: Academic Press. CHANGEUX, J. P., PODLESKI, T. R. & WOFSEY, L. (1967). Affinity labelling of the acetylcholine-receptor. Proc. natn. Acad. Sci. U.S.A. 58, CHANGEUX, J. P., KASAI, M. & LEE, C. Y. (1970). The use of a snake venom toxin to characterize the cholinergic receptor protein. Proc. natn. Acad. Sci. U.S.A. 67, CLELAND, W. W. (1964). Dithiothreitol. A new protective reagent for SH groups. Biochemistry, N.Y. 3, DEL CASTILLO, J., BARTELS, E. & SOBRINo, J. A. (1972). Microelectrophoretic application of cholinergic compounds, protein oxidizing agents, and mercurials to the chemically excitable membrane of the electroplax. Proc. natn. Acad. Sci. U.S.A. 69, DEL CASTILLO, J., EscoBAR, I. & GiJ6N, E. (1971). Effects of the electrophoretic application of sulfhydryl reagents to the end plate receptors. Int. J. Neurosci. 1, DEL CASTILLO, J. & KATZ, B. (1956). Biophysical aspects of neuromuscular transmission. Prog. Biophys. biophys. Chem. 6, DE ROBERTIS, E. D. (1971). Molecular biology of synaptic receptors. Science, N.Y. 171, EISENBERG, R. S., How-EL, J. N. & VAUGHAN, P. C. (1971). The maintenance of resting potentials in glycerol-treated muscle fibers. J. Physiol. 215, ELDEFRAWI, M. E., ELDEFRAWI, A. T. & O'BRIEN, R. D. (1971). Binding sites for cholinergic ligands in a particular fraction of electrophorus electroplax. Proc. natn. Acad. Sci. U.S.A. 68, ELMQVIST, D. & QUASTEL, D. M. J. (1965). Presynaptic action of hemicholinium at the neuromuscular junction. J. Physiol. 177, EPSTEIN, R. & GRUNDFEST, H. (1970). Desensitization of gamma aminobutyric acid (GABA) receptors in muscle fibres of the crab Cancer boreali. J. gen. Phys. 56, FELTZ, A. & MALLART, A. (1971). An analysis of acetylcholine responses of junctional and extrajunctional receptors of frog muscle fibres. J. Physiol. 218, FULpius, B. W., KLETT, R. P., COOPER, D. & REICH, E. (1972). The nicotinic acetylcholine receptor: Characteristics and properties of a macromolecule isolated from electrophorus electricus. Proc. of the 5th Int. Congress on Pharmacology, San Francisco. Abstracts of invited presentations, pp , GAGE, P. W. & McBURNEY, R. N. (1972). Miniature end-plate currents and potentials generated by quanta of acetylcholine in glycerol-treated toad sartorius fibres. J. Physiol. 226,

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