3,3-bi8[ac-(trimethylammonium)methyl]azobenzene (cis-bis-q) is photoisomerized by

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1 J. Phy8iol. (1983), 338, pp With 6 text -figure Printed in Great Britain ACTIVATION AND DESENSITIZATION OF ACETYLCHOLINE RECEPTORS IN FISH MUSCLE WITH A PHOTOISOMERIZABLE AGONIST BY M. M. WEINSTOCK From the Divi8ion of Biology, , California In8titute of Technology, Pa8adena, CA 91125, U.S.A. (Received 21 July 1982) SUMMARY 1. Voltage-clamped muscle fibres of the fish XenoMy8tus nigri8 were bathed in a solution containing 3,3-bi8[a-(trimethylammonium)methyl]azobenzene (ci8-bis-q; 100 nm). A flash of light photoisomerized some of the ci8-bis-q (non-agonist form) to tran8-bis-q (agonist form). This resulted in agonist-induced current. 2. Current noise of channels activated by both trans-bis-q and ACh was analysed to find the channel conductances and open times associated with these two agonists. Channels activated by tran8-bis-q and ACh had similar conductances (20-30 ps) and open times (3-4 msec) despite the photolabile azobenzene group of Bis-Q. 3. Light flashes subsequent to the first flash caused further increases in the ratio of tran8-bis-q to ci8-bis-q and accompanying increases in agonist-induced current. Eventually, agonist-induced currents levelled off as a photo-equilibrium state was reached with a constant tran8 cis ratio. 4. After the photo-equilibrium current level was reached, light flashes caused temporary increases in agonist-induced current which decayed back to equilibrium in seconds. This result is interpreted according to a model in which trans-bis-q molecules are bound to a subpopulation of desensitized receptors, preventing recovery to native receptor. A flash of light then converts some tran8-bis-q molecules bound to the desensitized receptor to the cis isomer. The newly formed cis-bis-q molecule may then unbind, allowing the desensitized receptor to recover. 5. When light was flashed on muscle exposed to nm-cis-bis-q, large ( na) agonist-induced currents were produced. These currents decayed exponentially over several seconds as the fibre desensitized. This result confirms that a first-order process underlies the onset of desensitization. INTRODUCTION A flash of light may be used to cause a rapid change of the concentration of agonist in the vicinity of cholinergic receptors. This occurs when the inactive compound 3,3-bi8[ac-(trimethylammonium)methyl]azobenzene (cis-bis-q) is photoisomerized by light to the agonist tran8-bis-q (Bartels, Wassermann & Erlanger, 1971; Lester & Chang, 1977) (Fig. la).

2 424 M. M. WEINSTOCK This method offers a number of advantages. First, one may apply a known concentration of agonist to receptors more rapidly than can be achieved by bath application methods. Secondly, a flash of light may perturb the structure of the agonist-receptor complex by photoisomerizing bound molecules of trans-bis-q, the active isomer, to the inactive cis isomer (Nass, Lester & Krouse, 1978). A NEN-NC L N+(CH3)3 B CH2 I N I(CH3)3 Cis-Bis-Q (non-agonist) CH2 ~ ~ ~~~~ NUNCH CH2 I N+(CH3)3 0 CH3 -C-0-CH2-CH2-N+(CH3)3 1C CH2 N+(CH3)3 Trans-Bis-Q (agonist) ACh Fig. 1. A, Bis-Q; the trans configuration of Bis-Q is an agonist; the Cis form is not. Cis -. trana and trana -. Cta photoisomerizations proceed at all wave-lengths between 300 and 500 nm. In white light, the photo-equilibrium state is 65 % trana and 35 % cia. B, ACh is shown for comparison. One goal of this study was to compare the elementary properties of the Bis- Q-activated channel to the ACh-activated channel. It is known that the open time of the activated channel varies with the nature of the agonist: suberyldicholine, for example, induces a longer mean open time than ACh, while the open time associated with carbachol is briefer (Colquhoun, Dionne, Steinbach & Stevens, 1975). It was therefore of interest to test whether the properties of the Bis-Q-activated channel differ from the properties of the ACh-activated channel. The results of these experiments indicate that channels activated by trans-bis-q have a similar open time and conductance compared to channels activated by ACh. A second goal was to study the nature of desensitization, the diminished response seen during continued exposure to agonist. The rate of desensitization onset depends on the concentration of agonist and the manner in which it is applied to the preparation. Ionophoresis (Katz & Thesleff, 1957), local perfusion (Manthey, 1966) and bath application (Adams, 1975 a) have been used to study desensitization in frog muscle. The light flash method was used here to apply a known concentration of agonist quickly to fish muscle receptors. Then the decline of agonist-induced current was followed as a measure of desensitization. Generally, the current decayed exponentially. This lends support to the hypothesis that a first-order process underlies the onset of desensitization. One hypothesis of desensitization proposes that desensitized receptors are hindered from recovery by tightly bound agonist (Katz &

3 ACh RECEPTORS IN FISH MUSCLE Thesleff, 1957; Anwyl & Narahashi, 1980). This notion is tested in this paper by using the light flash method to alter the configuration of bound agonist molecules. The results indicate that recovery from desensitization can in fact be accelerated by a light flash. METHODS 425 Preliminary experiments were performed on several species of fish, including goldfish, hatchet fish, and several members of the gymnotid family (since trans-bis-q is a poor agonist at frog or rat muscle). The African knife fish, Xenomystuw nijri8, was chosen for the experiments because it had muscles that withstood impalement well and were unusually sensitive to the agonist tran8-bis-q. A longitudinal muscle attached to the skin was used. Individual muscle fibres are 1-2 mm long with nerve terminals distributed all along the superficial and deep surfaces. The muscles were placed in one of two Ringer solutions. Usually, the fish were dissected in Electrophorus Ringer solution, containing NaCI, 160 mm; KC1, 2-5 mm; CaC12, 2-0 mm; HEPES, 10 mm, ph 7-2. For the actual experiments, the fish were placed in a solution containing NaCl, 116 mm; KCl, 2-0 mm; CaCl2, 2-0 mm; HEPES2-0 mm;tetrodotoxin, 2 x 1O-' M(toprevent contraction), ph Additionally, the muscles were often pre-treated with 0-2% collagenase (Sigma, Type I) to aid diffusion of agonist into the synaptic cleft. Gi8-Bis-Q, purified by h p.l.c., was added to the Ringer solutions. The preparation was either left at room temperature ( C) or was cooled to 6-10 C with a Peltier-effect thermoelectric cooler, The muscle fibres were impaled at their centre with a voltage-sensing micro-electrode (filled with 3M-KCI), and a current-passing micro-electrode (filled with 2M-K citrate); currents were recorded under voltage clamp. The distance from the middle to the end of the fibre was generally less than 1 mm so the entire fibre was under voltage clamp. For experiments with ACh, the drug was released from an ionophoretic pipette near a voltage clamped fibre. For experiments with Bis-Q, the muscle was bathed in Ci8-Bis-Q and light flashes were produced by a xenon flash tube. These flashes converted ci8-bis-q (the non-agonist configuration) to trans-bis-q (the agonist configuration). The optical set-up is described by Nass et al. (1978) with these differences: the flash tube was arranged above a compound microscope so that the flash passed through the microscope objective and was focused onto the muscle as a circle one to two millimetres in diameter. In this way, the one to two millimetre-long fibre under study was exposed to a concentration jump of tran8a-bis-q. In a solution containing pure cis-bis-q, a light flash causes a ci8 -trand conversion. In a solution containing a mixture of cis and trans both cis-+ trans and trans - cia conversions occur with a light flash. After several flashes, the photo-equilibrium state is reached where 65% of the solution is trans and 35% of the solution is cia (Lester, Krouse, Nass, Wassermann & Erlanger, 1980). To study the photochemical consequences of the flashes, a cuvette containing ci8-bis-q (82 FM) was placed in the apparatus at the usual position of the cell. Cis -. trans photoisomerizations were monitored by measuring changes in absorbance (Lester et al. 1980). The photo-equilibrium state was approached exponentially with the number of flashes. The flash had a photoisomerization potency of 0-58 flash-', that is, each flash drove the isomeric composition 44% of the way toward the photostationary state. Diffusion of agonist over small distances (microns) from the circle after the flash cannot be ruled out. However, it appears unlikely that large scale dilution of tran8-bis-q occurred due to diffusion from the circle on the time scale of the experiments because agonist-induced currents may be stable for minutes after a flash when low concentrations of Bis-Q are employed and desensitization is minimal (see Fig. 2A, first two flashes). Bis-Q current noise was collected during such plateaus. The resulting agonist-induced current was amplified and recorded on an FM tape recorder. Later, selected portions of the record were digitized and fed into a NOVA- 2 computer; current fluctuations were analysed using the autocorrelation function. Further details are given in Magleby & Weinstock (1980).

4 426 M. M. WEINSTOCK A of t 10 nal 10 sec 20 na 10 msec C 40 nal 50 msec D 20 nal 10 msec Fig. 2. Agonist-induced currents following light flashes in a voltage-clamped cell bathed in 100 nm-cis-bis-q. Inward currents are represented as downward deflexions. A, the increment of agonist-induced current diminishes as the photo-equilibrium state is approached and as desensitization develops. Flashes are marked by arrows. Voltage jumps are shown as vertical lines. By the third flash, the current increase has slowed considerably. By the fourth flash, a current decrease precedes the increase. B, response to a first flash on a faster time base. Flash artifact is now apparent (different cell than in A). C, response to a seventh flash. A current decrease followed by a slow increase is apparent (different cell). D, flash without agonist. Only flash artifact is seen (different cell).

5 ACh RECEPTORS IN FISH MUSCLE 427 RESULTS Elementary properties of the trane-bis-q-activated channel. In a voltage-clamped cell bathed in 100 nm-ci8-bis-q a light flash produces a concentration jump of trans-bis-q from about 0 to about 30 nm. There is an accompanying increase in the voltage-clamp current as nicotinic channels are activated (Fig. 2A). TABLE 1. Comparison of conductance and open time for channels activated by ACh and tranw-bis-q. (Mean+s.E. of mean) -75 mv, 10 IC ACh Tranw-Bis-Q y ps (n = 3) ps (n= 4) r 3'5 + 0O2 msec (n = 3) 3±6 1 msec (n= 4) A Trans-Bis-Q B ACh 1o <1020 <~ ' _' _' _' _' _' _' Time interval (msec) Time interval (msec) Fig. 3. Semilogarithmic plot of autocorrelation function of Bis-Q current noise (A) and ACh current noise (B). Channel open time is the time constant of decay of the autocorrelation function, which is 3-09 msec for the cell exposed to tran8-bis-q and 3-11 msec for the cell exposed to ACh. Mean single-channel current is equal to the intercept of the autocorrelation function divided by the mean agonist-induced current. The intercepts in (A) and (B) are the same and the mean agonist-induced currents for both cells were 25 na. Holding potentials -75 mv; 10 C. Analysis ofthese currents revealed that the elementary properties ofthe trans-bis-q activated channel are similar to those of the ACh-activated channel in spite of the azobenzene group which forms the bulk of the Bis-Q molecule and renders it photoisomerizable (Table 1). Fig. 3A shows the autocorrelation function of current noise collected from a fish muscle cell under voltage clamp (-75 mv, 10 'C) bathed in ci8-bis-q that had been exposed to a light flash. For comparison, Fig. 3B shows an autocorrelation function of current noise recorded from a muscle fibre to which ACh had been applied by micro-ionophoresis. In both cases, the total agonist-induced current was 25 na. The similarity of slopes and intercepts of these autocorrelation functions demonstrates that channels activated by tran8-bis-q and ACh possess similar conductances and open times. Given the similarity with regard to channel open times and conductances that has just been demonstrated for ACh and tran8-bis-q, it is curious that tranm-bis-q has a much lower KD than ACh in the eel electroplaque: tran8-bis-q, 200 nm; ACh, 20 /M

6 428 M. M. WEINSTOCK (H. A. Lester & R. E. Sheridan, unpublished). It may be that the rate of channel opening for a given agonist concentration is much greater for trans-bis-q than for ACh (Lester et al. 1980). General observations on responses to a flash. The increase in voltage-clamp current after a flash usually occurs in two phases. The rapid phase occurs on a time scale of milliseconds and the slower phase requires seconds (Fig. 2A and B). A second flash causes a further, though smaller, increase in current because the concentration of cis-bis-q was decreased by the first flash (Fig. 2A). Also, the increases in current associated with subsequent flashes are very much slower (third flash, Fig. 2A). After three to four flashes, the kinetics become complex and the current after a flash first decreases and then increases (Fig. 2A and C). With repeated exposure to white light, the Bis-Q concentration reaches a photostationary state where about 65 % of the molecules are in the trans state (agonist) and 350 of the molecules are in the cis state (non-agonist). This equilibrium point is reached after about six flashes. Beyond this point, no permanent increases in holding current are produced by further flashes; indeed, holding current often decreases somewhat as the fibre desensitizes. Speed of initial flash-induced relaxation. The time constant of the initial current increase following a flash is of the same order as the channel open time. After a sudden change in one of the factors determining receptor activation, the channel population is expected to move to a new equilibrium state. The time course of such a relaxation yields information about the rate-limiting steps at the receptor. In the present case, the number of open channels increases exponentially as a function of time until it reaches an equilibrium level. At the low agonist concentrations used, the current is expected to increase with a time constant equal to the average channel open time. This phenomenon was first noted by Lester & Chang (1977) in electroplaque and was not extensively studied here. Anomalous current movements after many flashes. The slow, anomalous current changes seen in Fig. 2 raise the following question: does the channel open or close more slowly as a result of repeated flashes or prolonged exposure to trans-bis-q? It was found that although the light-flash relaxation is altered, autocorrelation time constants of current noise were unchanged after many flashes, indicating that channels closed at the same rate. Further information was obtained with voltage-jump relaxations (Adams, 1975b; Neher & Sakmann, 1975; Sheridan & Lester, 1975). Fig. 4 shows that when membrane potential is jumped from -50 to -100 mv there is a two-phase increase in current: first, an instantaneous increase in holding current due to an increased driving force on the channels already open; then, an exponential approach to a larger current as more channels open. There are more channels open at equilibrium at hyperpolarized potentials because a, the closing rate, decreases in response to the voltage change (Magleby & Stevens, 1972). When the membrane potential is jumped back to -50 mv, there is an instantaneous decrease in holding current followed by an exponential decrease in current as the population of open channels diminishes. The agonist-induced current (corrected for the increased driving potential) increased after the hyperpolarization to the same extent as the time constant of the exponential relaxations. I-100/I-50 was (S.E. of mean, n = 5) compared to -100/T-i50 of (n = 5). Thus, the holding current increased proportionally to the channel lifetime.

7 ACh RECEPTORS IN FISH MUSCLE 429 These observations indicate no permanent change in the way receptors operate after many flashes. Therefore, the complex, slow current movements following a third or fourth flash remain to be explained. The patterns seen in Fig. 2A and C are not simply artifacts of the flash because they are not observed when there is no Bis-Q (Fig. 2D). It may be that a flash of light converts trans-bis-q which is bound to desensitized receptors to cis-bis-q, leading to a release of cis-bis-q into the synaptic cleft. Cis-Bis-Q is a channel blocker at micromolar concentrations (Nerbonne, Sheridan, Chabala & Lester, 1983). 40 nal 10 msec Fig. 4. Record of agonist-induced current during a voltage jump in a fibre exposed to trans-bis-q. Membrane potential was held at -550 mv and jumped to -I00 mv for 100 msec and then back again. Passive currents have been subtracted. Desensitized receptor binds agonist with very high affinity, 5-20-fold higher than native affinity, (Weber, David-Pfeuty & Changeux, 1975). If a portion of receptors in the cleft become desensitized with continued exposure to agonist, then large amounts of trans-bis-q could be bound to receptors lining the cleft. Presumably only trans-bis-q, the agonist form, would be bound to desensitized receptors. The effect of a light flash on this bound trans-bis-q would be to convert a portion of these molecules to the cis form (moving the bound molecules closer to the 65 % trams: 35 % cis photo-equilibrium state). Because the density of receptors in the cleft is so high (15,000/1u2, equivalent to 500 #m; Land, Salpeter & Salpeter, 1981), an appreciable concentration (micromolar) of cis-bis-q might build up in the cleft as a result of a flash. Thus, a light flash could convert some trans-bis-q bound to receptors to the cis configuration; the cis-bis-q could unbind from the desensitized receptors and then block open channels. With time, the cis-bis-q could diffuse out of the cleft. Thus, the complicated current traces seen in Fig. 2 could be easily explained by this hypothesis. A further consequence of this hypothesis is taken up in the next section. Desensitization in fish muscle. At low concentrations of Bis-Q (100 nm) and at temperatures between 6 and 10 'C, temporary increases in current are seen at the photo-equilibrium state; this phenomenon may be related to recovery from desensitization. At higher concentrations of Bis-Q and at room temperature, desensitization onset is more rapid and thus more easily studied. (a) Recovery from desensitization. At low concentrations of cis-bis-q (100 nm), the agonist-induced current reached a maximum after several flashes. At this point, presumably, the cis61±trans photo-equilibrium state was reached. Flashes of light after this point caused a transient decrease in current followed by a transient overshoot above the equilibrium level which decayed back to equilibrium (Fig. 5). The overshoot may represent a partial, temporary recovery from desensitization due to removal of agonist from desensitized receptor, allowing these receptors to be activated (see Discussion).

8 430 M. M. WEINSTOCK nal 15 sec Fig. 5. Overshoot of current after the solution has reached photo-equilibrium. Agonistinduced current following flashes six to ten are shown. Base line indicates starting current. Holding potential, -50 mv; 10 'C. Different cell than that shown in Fig. 2. Note that agonist-induced current tends to decrease with time, indicating desensitization. A B 40 na[ 30 sec C 40 na 60 sec Time (sec) Fig. 6. Desensitization in fibres bathed in ci8-bis-q: agonist-induced currents following a first flash. Holding potential, -50 mv; C. A, 300 nm-bis-q. B, semilog plot of current decays in 300 nm-bis-q (0) and 600 nm-bis-q ( x ). C, 300 nm-bis-q. Note slight 'bump' after current decline.

9 ACh RECEPTORS IN FISH MUSCLE (b) Onset of desensitization. Desensitization was usually seen to some extent with all concentrations of Bis-Q used ( nm). Desensitization was most evident, however, at the concentrations > 300 nm. The first flash converts about 30 % of the cis-bis-q to the trans isomer. This amounts to a concentration jump from about 0 to 90 nm-trans when the starting concentration is 300 nm-cis. Following the flash, the agonist-induced holding current increased to a peak of about na, then decayed exponentially with time (Fig. 6A). When the muscle was bathed in 600 nm-cis-bis-q, a flash likewise jumps the trans-bis-q concentration to about 180 nm. The onset of desensitization followed an exponential time course but the time constant was roughly halved (Fig. 6B); Tdecay, 300 nm was sec (S.E. of mean, n = 3) and Tdecay, 6oo nf was sec (S.E. of mean, n = 2). In one cell, the agonist-induced current decayed as two exponential. Feltz & Trautmann (1980) have made similar findings in frog neuromuscular junction. Also, in fish muscle fibres cells that had undergone previous exposure to trans-bis-q, current decayed unusually slowly, in minutes rather than seconds. 431 DISCUSSION Light can photoisomerize cis-bis-q to trans-bis-q which is a cholinergic agonist. Trans-Bis-Q combines with ACh receptors to open channels, leading to a post-synaptic current. Analysis of the resulting current noise indicates that the conductance and open time of channels activated by trans-bis-q and ACh are similar. This is an interesting result in view of the azobenzene group of Bis-Q which gives it a structure quite different from ACh. The similarity of the ACh- and trans-bis-q-activated channel encourages the use of Bis-Q to study normal channel operation. Conversely, the slow, complex current changes seen after many flashes are not typical of ACh receptor behaviour. Fast phase of relaxation following the first flash. The first relaxation after a flash seemed similar to the relaxation found in flash experiments with Electrophorus. This phenomenon was interpreted by Lester & Chang (1977) to represent the equilibration of agonist with its receptor, the time constant of relaxation equalling the reciprocal of the sum of the closing and opening rates, 1/(ac+6). Slowphase ofcurrent relaxationfollowing thefirstflash. The increase ofagonist-induced current in response to the first flash had a slow component (seconds). This phenomenon may be related to the slow-current rises seen after many flashes. Alternatively, there may be a long diffusion path to the receptors on the deep surface of the muscle fibre. On the deep surface, there may be no more than a micron or so of space between fibres. At a concentration of 30 nm-trans-bis-q, there are only 18 molecules per cubic micron. It may take seconds for the agonist molecules to equilibrate with their receptors on the deep surface because agonist molecules must diffuse along a path of tens of microns around the circumference of the fibre before they reach a sufficient concentration in the vicinity of the synapse. Desensitization onset. There are two phenomena often associated with desensitization: its onset is exponential and its rate depends on the concentration of agonist (Adams, 1975 a; Katz & Thesleff, 1957; Lester, Changeux & Sheridan, 1975). The light flash method of delivering agonist generally confirms the results seen with other

10 432 m. m. WEINSTOCK methods: there is a simple exponential decay in current; the rate of this decay is a function of agonist concentration. Desensitization recovery. The hypothesis considered here is that desensitized receptor has agonist bound to it; when agonist unbinds, the receptor may recover quickly. The light-flash technique provides a way to convert an agonist molecule to a non-agonist molecule even while it is attached to the receptor, allowing the hypothesis to be tested. After several flashes there is no permanent increase in voltage-clamp current, but after the transient decrease that follows a flash there is a transient increase in current above equilibrium levels. This transient increase is even more striking after a series offlashes spaced only sec apart (flashes eight nine and ten of Fig. 5). It appears, therefore, that the effect of a flash in causing recovery may be potentiated by a flash occurring shortly before: compare the recovery caused by flashes nine and ten to the recovery caused by flashes seven and eight. This may indicate that two agonist molecules are bound per desensitized receptor-channel complex and that recovery occurs when both agonist molecules dissociate. In simple terms, the first flash of a series may often dislodge only one agonist molecule of a pair while the second and third flashes dislodge the remaining agonist molecule and are therefore more effective. This hypothesis could be explored further by noting the effects of flash intensity; Bumps. Adams (1975 a) noted 'bumps' in the holding current following wash-out of a depolarizing drug; he speculated that agonist released from desensitized receptors was activating undesensitized receptors. Bumps were seen infrequently in the experiments reported here (Fig. 6C). Because these bumps come about while agonist is still present and not during a wash-out of agonist, it is unlikely that they represent a recovery phenomenon in the experiments reported here. Perhaps the bumps are simply a consequence of a decrease in agonist-induced current. A simple explanation may be that Ca2+ enters the fibre during the rising phase of the peak; this could cause slight tension to appear in the fibre, leading to a relatively small deflexion of holding current that might not show up on the rising phase. When inward current diminishes, the tension may relax as the internal free [Ca2+] decreases, leading to the artifactual bump. Comparison offish muscle to eel electroplaque. Under certain conditions, Nass et al. (1978) found a rapid ( < 1 msec) decrease in current following a flash; this phenomenon was referred to as phase 1. A later, slower decrease was also seen and was referred to as phase 3. However, current after a light flash did not decrease rapidly in fish muscle; there was only a relatively slow (tens of milliseconds) decrease in current resembling 'phase 3.' Nass et al. (1978) showed an overshoot of current following a flash in Fig. 4a and b and Fig. 12 of that work which may be analogous to the overshoot shown in this paper in Fig. 5. I thank Dr H. A. Lester for support and advice, Drs B. Erlanger and N. Wassermann for supplying this lab with Bis-Q, Dr Jeanne Nerbonne for purifying cis-bis-q and Drs K. Magleby and J. Barrett for criticism of the manuscript. This study was supported by N.I.H. grants NS and 1F32 NS

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