independent causes but are coupled: in given conditions, the onset can be essentially fast, and the recovery slow.

Transcription

1 J. Physiol. (1982), 322, pp With 6 text-figures Printed in Great Britain DESENSITIZATION AT THE FROG NEUROMUSCULAR JUNCTION: A BIPHASIC PROCESS BY ANNE FELTZ* AND ALAIN TRAUTMANN From the Laboratoire de Neurobioloqie, Ecole Normale Supe'rieure, 46, rue d'ulm, 7523OParis Cedex 5, Fiance (Received 6 May 1981) SUMMARY 1. The desensitization of the cholinergic receptor has been investigated at the frog neuromuscular junction. The agonist was either perfused or applied by ionophoresis. 2. In all situations, desensitization develops in two phases: a fast one, experimentally in the second range but likely to be briefer, and a slower one, which extends over tens of seconds. 3. When the presence of the agonist is prolonged, desensitization approaches a steady state, estimated through the amplitude of a test response. In steady-state conditions, this amplitude depends upon the desensitizing agonist concentration. The dose-response curve for desensitization induced by carbachol (CCh) indicates that half of the receptors can be desensitized at room temperature in the presence of 2-3 /LM-CCh. The shape of the curve suggests that one desensitized receptor can bind two CCh molecules. 4. The recovery from desensitization, estimated with a repetitive test pulse, displays two exponential phases. The time constant of the fast phase is sec, and 4-5 min for the slow phase, regardless of the concentration or the nature of the agonist (acetylcholine or carbachol). 5. The factor which most strikingly affects the relative amplitudes of the fast and slow phases of recovery is the duration of the (desensitizing) agonist application. Desensitizations lasting a few seconds are followed by a 'fast' recovery, whereas the slow phase of recovery is prominent when the agonist has been applied for more than 2 min. 6. The fast and slow phases of desensitization onset and offset are not due to independent causes but are coupled: in given conditions, the onset can be essentially fast, and the recovery slow. 7. All our findings can fit in a cyclic scheme of desensitization, derived from the one of Katz & Thesleff (1957) with two modifications: whether activatable or desensitized, one receptor molecule would have two agonist binding sites; moreover, the desensitized receptor would exist in two distinct and interconverting conformations: D1, giving rise to the fast phases of onset and offset, and D2, responsible for the existence of the slow components of desensitization. * Present address: Laboratoire de Physiologie Respiratoire, C.N.R.S. 23, rue Becquerel, 6787 Strasbourg, France. 9 PHY 322

2 258 A. FELTZ AND A. TRAUTMANN INTRODUCTION In many biological systems, when receptors are submitted to a prolonged exposure of an agonist, the response is not maintained throughout the duration of the exposure but decreases with time. At the nicotinic receptor, it is thought that in presence of agonist, the receptor undergoes a conformational change, from a resting, activatable state, to a non-conducting desensitized form (Katz & Thesleff, 1957). The desensitized receptor binds the agonist with high affinity (Weber, David-Pfeuty & Changeux, 1975; Weiland, Georgia, Wee, Chignell & Taylor, 1976). The time course of the transition from the activatable to the desensitized state has generally been described as a single exponential process. However the values reported for the time constant vary markedly with the technique employed. When the agonist is ionophoretically applied, desensitization onset and recovery are described by much faster kinetics than when desensitization is obtained by agonist superfusion. To consider for example recovery, in the first case, the time constant of recovery is in the order of 1 sec (Katz & Thesleff, 1957; Magazanik and Vyskocil, 1975); in the second case, it varies between 2 and 45 sec (Rang & Ritter, 197; Scubon-Mulieri & Parsons, 1977). We have recently observed that desensitization onset is not a monophasic but a biphasic process which can be kinetically described by the sum of two exponentials (Feltz & Trautmann, 198). Anwyl & Narahashi (198) and Clark & Adams (1981) have made similar observations at the frog neuromuscular junction. Moreover the cholinergic receptor which induces a chloride permeability in molluscan neurones and displays pharmacological characteristics close to the end-plate receptor (Tauc & Gerschenfeld, 1962), also presents a biphasic desensitization (Andreev and Vulfius, 198). Here, we confirm the biphasicity of the onset of desensitization, using various techniques. We further show that the recovery from desensitization is also a biphasic process. The aim of the present work is to characterize some of the kinetic parameters of these four steps of desensitization and to propose a simple kinetic model compatible with our findings. This model, which assumes two distinct conformations of the desensitized receptor, accounts for most of our observations. Similar proposals arise from the flux studies of Neubig & Cohen (198) and the single channel analysis of Sakmann, Patlak & Neher (198). These results have constituted part of a thesis (Trautmann, 198). METHODS The experiments have been performed on the voltage clamped fibres of cutaneous pectoris of Rana emculenta, at room temperature (2-24C). The muscle was stretched in a 1 ml chamber and the synaptic areas located through Nomarski optics using a water immersion objective lens (total magnification x 4). For superfusion, a jet of Ringer was directed onto the synaptic area and the rate adjusted either to a low value when the aim was to avoid any mechanical disturbance (2 ml/min) or at a rate of 15 ml/min to obtain fast concentration changes. The voltage and current electrodes were filled with 2 m-kcl and 2 M-K acetate respectively. To voltage clamp the muscle fibre, the feedback gain (usually x 15) was adjusted at each voltage so that the mepps reduction was > 95 %. A Bessel filter (48 db/decade) was generally used (setting range: 9-2 Hz). Current was monitored by a current to voltage converter (virtual ground). Holding potentials were between -7 to -1 mv. The Ringer's solution had the following composition (in mm): NaCl, 115; KCl, 2; MgCl2, 5; Hepes,

3 DESENSITIZATION AT THE END-PLATE 259 2; and ph was adjusted to 7-4. Ca2+ was omitted from this Ringer's solution (and replaced by Mg2+) for two reasons: first, the entry of Ca2+ into the cell through ACh-activated channels can produce local contractions and, thus, reduce the stability of the recording; second, a Ca-free Ringer is necessary to evoke focal e.p.c.s (see below). Usually acetylcholinesterase (AChE) was irreversibly inactivated by exposing the muscle for 1 hr at room temperature to 4 x 1-7 M-O-ethyl-S2 diisopropyl amino-ethyl-methane-thiophosphonate (MTP). MTP was a gift of Dr Leterrier (Vigny, Bon, Massoulie' & Leterrier, 1978). Desensitization was either induced by acetylcholine (ACh, Sigma) or carbachol (CCh, Sigma). The drugs were either applied by superfusion at the indicated concentration or by ionophoresis from pipettes filled with ACh 1 M or CCh 5 M. The amount of desensitization has been estimated from the reduction in amplitude of a test response. In most cases this response was due to transmitter release from nerve terminals: we have used miniature end-plate currents (m.e.p.c.s) or nerve-evoked focal end-plate currents (e.p.c.s). In the latter case, the nerve was stimulated in the Ca free Ringer; transmitter release was only evoked under the tip of a Ca pipette, where the local Ca concentration was raised (Katz & Miledi, 1965). Occasionally, the test response was evoked by CCh ionophoresis. The data were stored on a tape recorder (Racal, Store 4) and slowly replayed onto a pen recorder (Brush 28). RESULTS The onset of desensitization Fig. 1 shows in three distinct desensitizing conditions, that the decrease of the agonist-induced current occurs in two exponential phases: one with a time constant in the second range, the other with a time constant of a few tens of seconds. In Fig. 1 A the agonist is applied by brief repetitive ionophoretic pulses. The amplitude of the resulting response is stable when the pulse interval is long enough (8 sec in the case illustrated). When the pulse interval is decreased to -6 sec the amplitude of the response declines with time along two exponential phases, as indicated in Fig. ID, with time constants 1-1 and 9-3 sec respectively. One can also observe in Fig. 1A that, after the pulse interval has been increased again, a rapid but incomplete recovery of desensitization is followed by a second, slower phase of recovery. This point will be examined in more detail later. In Fig. 1 B, a single, prolonged ionophoretic pulse of carbachol leads to an inward current which decreases first rapidly and then more slowly. During the first minute ofapplication, the kinetics ofdesensitization can be well described by two exponential, with time constants 1-3 and 42 sec (Fig. 1 E). We show another example of the biphasic nature of desensitization in Fig. 1 C, where solutions containing 4 and 1 #M ACh were applied successively at the same end-plate. In the presence of 1 1M-ACh the current decay is described by two exponential with time constants of 2-3 and 67 sec (Fig. IF). Note that in this experiment, the AChE had not been inactivated. As a consequence, practically no ACh reached the receptors facing the nerve terminal, protected by the AChE and thus, did not desensitize them: this explains why in this experiment the amplitude of the m.e.p.c.s is not affected by the desensitizing agonist (cf. Feltz & Trautmann, 198). The three cases illustrated in Fig. 1 display biphasic onsets of desensitizations. However, depending on the way the agonist was applied, the decline can appear to be monoexponential, as some conditions strongly favour one phase or the other. For instance, the two phases of desensitization are clearly seen during the application of 1,sM-ACh. However, at the lower agonist concentration (4 SM), the first component 9-2

4 26 A. FELTZ AND A. TRA UTMANN is small and more difficult to observe. On the other hand, the fast initial phase can be missed (even at high agonist concentration) if the perfusion is too slow (see e.g. Fig. 2). Conversely, when the agonist is applied by ionophoresis (whereby a high local concentration of agonist is rapidly established), desensitization can appear as essentially fast. Other experimental conditions can give rise to time courses of desensitization that appear more complex than a dual exponential process. A detailed analysis of Fig. 1 B, for example, shows a late very slow phase of decay following the two early exponential H7~~~~~TIJ~~~L ~1 A 3 S. ~ 2-\ ~~~~~~~D l sec U Nt B : E 4O i ~~ ~~< -9 2 ~~~~~~~~ 3 sec 1 e k.< < 4-1 C C ~~~~1 F 5 sec Time (sec) Fig. 1. Biphasic onset of desensitization. Desensitization was produced at room temperature either by repetitive pulses of CCh (A), by a single long pulse of CCh (B) or by fast The corresponding decays are bath application of 4 and 1 1sM-ACh successively (C). plotted in semi-logarithmic coordinates in the right column. The fast components (hollow circles) are obtained after subtraction of the slow components from the first points. The slow component is always quantified with some uncertainty since steady state was not reached. In D, the pulse amplitude is assumed to approach an equilibrium value of 2 na. The two time constants are 1-1 and 9-3 see respectively. In E, the steady state is assumed to be zero. The time constants are 1-3 and 42 sec. In F', only the decay of the current induced by 1 1sM-ACh is plotted, assuming a zero amplitude steady-state value. The time constants are 2-3 and 67 sec, respectively. The holding potentials were -1 mv (A), -8 mv (B), -7 mv (C).

5 DESENSITIZATION AT THE END-PLATE 261 phases. This may correspond to a third phase of desensitization but we favour a simpler interpretation based on the fact that during ionophoresis, the agonist concentration is not homogenous. The observed response results mostly from the activation of receptors close to the tip of the ionophoretic pipette, and subjected to the highest agonist concentration. More distant receptors, submitted to lower agonist concentrations contribute, nevertheless, to a fraction of the response and this fraction is likely to change with time since the agonist will slowly diffuse. Despite the various incidences of the effects of experimental protocol on the observed rate of desensitization, the biphasic decline illustrated in Fig. 1 appears to be characteristic of the desensitization process. Steady-state desensitization When the agonist application is sufficiently prolonged, desensitization tends towards a steady-state. It is of interest to determine the dose-response curve for desensitization, i.e. to measure the fraction of receptors desensitized in steady-state conditions, as a function of agonist concentration. As a matter of fact, from the shape of this curve, one can estimate the number of agonist molecules bound per desensitized receptor molecule (see Appendix). In practice it is easier to measure the fraction of receptors not desensitized, i.e. still activatable. For this purpose, one cannot simply rely upon the decay of the response during a perfusion, because the peak of the response will already be affected by rapidly developing desensitization; we thus followed the decline of a fast test response during the application of the desensitizing agonist (see Feltz & Trautmann, 198). In order to be able to detect the response even when a large fraction of the receptors was desensitized the control amplitude had to be very large, and this led us to use in most cases the focal e.p.c. as a test response (see Methods). Furthermore, to avoid mechanical disturbances which could artefactually affect the amplitude of the e.p.c. the rate of perfusion was set at a low level, and as a consequence, the initial phase of desensitization was usually masked. In such conditions, we could proceed over minutes to reliable measurements of the amplitude of the evoked response. This point is shown in Fig. 2: the amplitude of the control response before CCh application was 375 na, a 1,sM-CCh application reduced it to 25 na within 1 min. This value seems close to a plateau one. It is possible to fit the whole decrease of the CCh induced current by an exponential of r = 135 sec, with a plateau value of 2 na, i.e. 5-3 % ofthe control. When the recording remained stable for 2 min following the end of the CCh perfusion, an almost complete recovery was observed on several occasions; this suggests that an eventual fatigue of the nerve terminal was not severely biasing our results. M.e.p.c.s amplitudes were used as the test response mainly at concentrations ofcch below 5 /M, because the expected changes are small and slow to appear, and thus the stability of the control had to be very reliable. In a few experiments we used both m.e.p.c.s and e.p.c. as test responses, and found very comparable results. This eliminates one objection to the use of nerve evoked responses, namely the possible occurrence of presynaptic effects during agonist application. In Fig. 2, a residual response is observed even after prolonged application of an agonist: this means that when desensitization has reach an equilibrium, a fraction of the receptors is still activatable.

6 262 A. FELTZ AND A. TRA UTMANN If R/R... is the ratio of the steady-state number of activatable receptors in the presence and in the absence ofcch, (1 - R/Rmax) gives the fraction ofnon-activatable, i.e. desensitized receptors, in the same conditions. Fig. 3 gives the dose-response curve for desensitization. One can see that Rmax/R increases more than linearly with the agonist concentration. A linear relation is obtained by plotting (Rmax/R-1)1 as ordinate. An explanation for this relationship will be presented in the appendix. 5-2 _. 1 _ 5 2 ~~~~~ ~~~~~~~~~~ 1 \ o Time (sec) Fig. 2. Development of a prolonged desensitization. The fraction of activatable receptors was evaluated by following the amplitude of a focal e.p.c. Each point represents the mean of 4 e.p.c.s, evoked at -1/sec and measured at-9 mv. Before the CCh perfusion, the control amplitude is 375 na (top arrow). At time, 1,um-CCh was perfused. The assumed final equilibrium (lower arrow), not reached, was subtracted from the original data (filled circles) to give the open circles. In the semilogarithmic coordinates used, an exponential decline is approximated (time constant 135 sec). Half of the receptors are desensitized for a CCh concentration of 2-3 PEm. A comparable value (3-5,um) can be interpolated from the results of the flux study of Eldefrawi, Aronstam, Bakry, Eldefrawi &; Albuquerque (198) (see e.g. their Fig. 2). Recovery from desensitization Time constants of recovery. The biphasic nature of the recovery process is illustrated in Fig. 4. Two episodes of desensitization obtained successively on the same cell are illustrated. Two independent CCh pipettes were used, one to deliver brief repetitive test pulses, the other one to deliver a prolonged desensitizing pulse. The instantaneous amount of desensitization was expressed as the ratio (-I)I-I,)I is the amplitude of the test response before agonist application (control), It its amplitude

7 DESENSITIZATION AT THE END-PLATE 263 at time t, 4. at time for recovery, i.e. at the end of agonist application, when washout was started. The ratio gives the reduction in amplitude of the test response, relative to the maximum reduction. A semi-log plot of (I -It)/(Ic-I) versus time in Fig. 4 shows clearly a biphasic recovery. In the first episode, the two time constants are 1-7 and 33 sec respectively. In the second episode, the slow phase of recovery can be described with the same time constant (33 sec); the short time constant is 5 see (the last value is smaller in this experiment than generally measured in similar conditions) E 2 1b/~~~~ 5 1 Carbachol concentration (MM) Fig. 3. Steady-state desensitization. Relation between (Rmx/R -1) I and CCh concentration. R/Rmax represents the fraction of activatable receptors relative to its maximum value, and measured in experiments similar to the one illustrated in Fig. 2. The standard deviation is indicated when at least four experiments were performed at the same CCh concentration. Points without S.D. bars represent individual experiments. When the CCh concentration equals 2-3 /M, R/Rmax = 5, i.e. half of the receptors are desensitized. The kinetics ofthefast phase ofrecovery seem independent ofthe nature of the agonist. When desensitization was induced by CCh, the recovery took place with a time constant of sec (mean + S.D. of the mean, n = 13 experiments). After an ACh induced desensitization, this time constant was 11I sec (n = 1). It is interesting to note that these values for the time constant of recovery from desensitization are quite close to those reported by Katz & Thesleff (1957), and Magazanik & Vyskocil (1975) for ACh, and by Adams (1975) for CCh (between 7 and 15 sec). Because it is so brief, the fast recovery could only be studied after ionophoretically induced desensitization, and thus the effect of agonist concentration was not quantitatively studied. Nevertheless, the estimated rate of the fast recovery was the same whether the desensitization was evoked with a very high dose of CCh (leading to complete desensitization in a couple of seconds) or with a moderate dose (where desensitization develops in tens of seconds). This suggests that the rate of fast recovery is independent of agonist concentration.

8 264 A. FELTZ AND A. TRAUTMANN The late phase of recovery could be studied after ionophoresis as well as after bath application of the agonist. Measurements of the time constant of the late phase of recovery lead to an overall estimate of 4-5 min, whatever the way desensitization had been induced. The estimate after ionophoresis ofcch was sec (mean + S.D., n = 7) and was sec (mean+s.d., n = 8) after perfusion of CCh (3-1/M). This mean value is in fairly good agreement with the values of 2 and 42 sec respectively, observed by Rang & Ritter (197) and Scubon-Mulieri & Parsons (1977). 8R 1 5 or A\ Time (sec) 5 1 IF I 15 2 I u -Z u Z j S 5 3 I Ii, F I..8 :- '11M IP'lli 1! 1: T, " 5 na K 3 sec Z 2 I1- -1 _ 1 5 L I Time (sec) 15 2 Fig. 4. Biphasic recovery from desensitization. Two episodes of desensitization, performed at -8 mv and 23 C on the same cell, are illustrated. Carbachol was applied by two independent pipettes, one delivering brief repetitive test pulses of agonist, and the other one delivering a continuous dose (during black bars). During the recovery, the test pulses were stopped from time to time to reduce a possible local accumulation of carbachol. The semi-logarithmic plots illustrate the time course of recovery: see text for the meaning of the ordinates. Time zero (for recovery) is indicated by the asterisks. Top: with a low dose of CCh, after a desensitization lasting 74 see and reducing the test amplitude to approximately one fourth, 55 % of the recovery develops with a brief time constant (1-7 sec); the long time constant has a value of 33 sec. Bottom: with a higher dose of CCh, after a desensitization lasting 13 sec and reducing also the test response to one fourth of the control, 87 % of the recovery is rapid (time constant 5 sec). The slow phase can ne described with the same time constant as the previous slow recovery (top).

9 DESENSITIZATION AT THE END-PLATE 265 The slow part of the recovery following a prolonged ionophoresis of ACh occurred with a time constant of sec (mean+s.d., n = 9). These values are compatible with the hypothesis that the recoveries after CCh or ACh-induced desensitizations have similar kinetics, though more experiments are needed to rule out small differences. 1 A o * 5 1NP~~~~~~~ o 5 1 QL Amplitude of residual response (%) CU ' 1 _ B ~. E _, 5 _ 8 * ~~~~ 5~~~~~ 1 2 Duration of agonist application (sec) Fig. 5. Effect of the previous (CCh induced) desensitization on the amplitude of the fast phase of recovery. This amplitude was estimated by extrapolation of plots similar to the one of Fig. 4 (see text for more details). A, effect of the extent of desensitization on the amplitude of the fast recovery. The abscissa give the amplitude of the residual response (just before the recovery starts), relative to the control response in percent. Filled and open circles represent, respectively, the responses obtained after a desensitization lasting more (@) or less () than 1 min. B, effect of the duration of the (desensitizing) agonist application on the amplitude of the fast recovery. Experiments where the test response was reduced to less than 2 % are illustrated by filled circles. Open circles are used when the test response was greater than 2 % of its control value. The curve is the graph of the function Rf = 1 exp (-t/1), (see Discussion). Amplitudes of the two phases of recovery. The two episodes of desensitization illustrated in Fig. 4 both reduced the test response to about 25 % of the control value. In the first case, the recovery was mainly fast, whereas in the second case, the slow phase was prominent. The two runs differed in the CCh concentration applied, and in the duration of the agonist application. In order to find out which parameter was

10 266 A. FELTZ AND A. TRAUTMANN controlling the kinetics of recovery a quantitative analysis was performed. The amplitude of the initial phase was estimated in each experiment from the semi-log plot of (I, -It)/(I'c-IO versus time. The amplitude of the slow phase (x %) was first obtained by extrapolation of the slow exponential component to time zero. The amplitude of the fast initial phase Rf was then calculated as (1-x) %, and its variations were followed in different desensitizing conditions. The effects of the depth of the previoub desensitization (expressed as the amplitude in per cent of the residual 3 R 2-1L Time (sec) Fig. 6. The fast and slow phases of desensitization are not independent of each other. The amplitude of a test response to CCh ionophoresis is plotted as a function of time (stimulation frequency: -2 Hz). Between the arrows, a steady dose of CCh was applied through a second pipette, and induced both a slight potentiation (transiently visible at the beginning of the CCh application) and a marked desensitization. The smooth line represents the sum of the two exponential for both the onset and for the offset. The two equilibrium values which are assumed, since not reached, are na (complete desensitization) for the onset and 24 na (control value) for the offset. The time constants are 23 and 135 sec for the onset and 15 and 3 sec for the offset. The fast component constitutes 71 % of the onset, but 25 % of the offset. response at the end of the agonist application) were first examined, and the data are summarized in Fig. 5A. No correlation is apparent. A large fast initial recovery can be observed after a slight or a deep desensitization, and similarly a small rapid initial phase is not related to the extent of the previous desensitization. On the other hand, when 'brief' desensitizations (lasting less than 1 min) are distinguished from the 'long' ones, two distinct clusters of points appear, suggesting that the amplitude of the fast phase of recovery is related to the duration of the previous exposure to the desensitizing agonist. This correlation is confirmed in Fig. 5B, where the relative amplitude of the fast recovery is plotted against the duration of the previous desensitization. One can see that, the shorter the exposure to an agonist, the larger the relative amplitude of the fast recovery. On the other hand, for exposures which had lasted longer than 2 min, recovery occurs mainly through a slow phase. This relationship does not depend on the depth of the desensitization. The results have been divided in two groups: those obtained after a large amount ofdesensitization (below 2 % of the control response) and those obtained after a smaller extent of desensitization (when the test response did not decrease below 2% of the control). The two series of points are not distinct from each other.

11 DESENSITIZATION AT THE END-PLATE 267 The fast and slow phases of desensitization are not independent of each other. From the results presented above, one can already conclude that the fast and slow components of desensitization are not two independent processes. If they were independent of each other, the relative amplitude of the fast component of the onset should be equal to the relative amplitude of the fast component of the recovery. Experimentally, this is generally not the case. This point is made obvious by the experiment illustrated in Fig. 6, which shows the evolution of a test response during desensitization and recovery. One can observe that both onset and recovery are biphasic processes (with respective time constants of 23 and 135 sec for the onset, and 15 and 3 sec for the recovery). The important point is that in this experiment the fast component constitutes 71 % of the onset but only 25 % of the offset (respectively 29 and 75 % for the slow component). This type of observation argues against a class of models where the two components of desensitization would be independent of each other. An example of such an 'independent' model which cannot be retained is the one proposed by Andreev & Vulfius (198): these authors suggested that the two components of desensitization could be related to two subpopulations of receptors, one desensitizing rapidly, the other one slowly. DISCUSSION During maintained application of a nicotinic agonist at the neuromuscular junction, the decline of the response appears to proceed in two phases, one of which develops in a few seconds, whereas the second one is extended over tens of seconds. Depending on the conditions used to apply the agonist, one of the two phases can become prominent. If the rate of access of the agonist to the receptors is slow, the fast phase can be obscured. On the contrary, high concentrations of the agonist reduce the relative amplitude of the slow phase and can make it negligible. Therefore, when high agonist concentrations are rapidly applied with ionophoresis, it may be possible to observe only the fast phase of desensitization; by contrast, with low agonist concentrations and slow perfusions one may well overlook the fast phase of desensitization. This might explain why, until recently, desensitization onset was described as a monophasic process. Recovery of desensitization also proceeds along two distinct phases, with time constants of about 1 see and 4-5 min. None of four factors which have been studied seem to affect the time constants of these two phases; these include the nature of the agonist (CCh versus ACh), the agonist concentration, the fraction of receptors one can still activate at the end of an agonist application, and, finally, the duration of the agonist application. But on the other hand, the relative amplitudes of the two phases are notably affected by this latter parameter. This observation may explain the wide scatter of the values previously reported for the recovery process. In experiments using ionophoretic applications the desensitization was usually brief and the recovery proceeded mainly along its fast component; in experiments using bath applied agonists the exposure was usually longer and the recovery largely slow. What kind of scheme can take into account the different phases of desensitization and recovery we have observed? It has been shown above that a suitable scheme has

12 268 A. FELTZ AND A. TRAUTMANN to take into account the absence of independence of the fast and slow components. Another constraint for this scheme will be its cyclic characteristics. To describe the kinetic characteristics of desensitization, Katz & Thesleff (1957) have proposed a cyclic model which is the simplest one taking into account a series of observations, including: (1) The fact that the onset of desensitization may (in some conditions) be slower than the recovery and (2) the fact that the rate of recovery is independent of the nature of the agonist. In the scheme, (see Appendix) if the binding of the agonist is much faster than the conformational changes, both onset and recovery will be monoexponential. But desensitization might become biphasic if, for example, the rate of dissociation of the agonist from the high affinity (desensitized state) was slow enough. Nevertheless (see Appendix) such a model is still not compatible with our observations. Our results are consistent with a cyclic model postulating the existence of two distinct desensitized forms, D1 and D2, instead of only one. The fast desensitization would correspond to the isomerization of the receptor from the R to the D1 form. The evolution towards D2 would appear as the slow process. The fast recovery would correspond to D1 - R and the slow one to D2 - R (or D2 - D1 - R). A brief desensitization where only the D1 state is reached, would thus be followed by a fast recovery. After a longer desensitization, both fast and slow components would be visible. After a long desensitization, the recovery will be essentially slow if most of the desensitized receptors are in the D2 form, i.e. if the affinity of the agonist for D2 is much higher than for D1. Fig. 5B, which gives the amplitude of the fast recovery as a function of the duration of the previous desensitization, can also be used to describe the rate of appearance of the D2 form, i.e. the rate of the slow phase of onset. A time constant of 1 sec (see Fig. 5B) is in the range observed for the slow onset of desensitization evoked by ionophoresis. This value is obviously a mean one, as the rate of slow onset increases with the agonist concentration (Feltz & Trautmann, unpublished). The existence of two distinct desensitized states has also been recently proposed by Sakmann et al. (198). These authors have observed that in presence of a high agonist concentration, the elementary channels observed in patch-clamp open in bursts, and these bursts are grouped in clusters. In our terminology, a burst would stop when the receptor changes from the R to the D1 state; a cluster would finish after a transition to D2. The fraction of time a channel is activatable or open (i.e.: bursting) in these experiments seems very high (with regard to the high agonist concentration, 2 /LM-ACh) when compared to the fraction of activatable receptors we have measured during steady-state desensitization. One explanation for this discrepancy could be that Sakmann et al. were not observing only one channel (for example, each cluster could correspond to a different channel). Biochemical studies (Neubig & Cohen, 198; Heidmann & Changeux, 198) have also recently led to a model in which the cholinergic receptor could exist in three different states of affinity: two of them, the 'high' and 'low' affinities were previously thought to correspond to the desensitized and activatable receptor respectively. It appears now more likely that these two states are two different desensitized states (our D2 and D1 states), and that the activatable receptor has an even lower affinity for the agonist, difficult to estimate in binding studies, precisely because of desensitization.

13 DESENSITIZATION AT THE END-PLATE 269 So, it now appears that the cyclic scheme of desensitization of Katz & Thesleff has to be extended first by postulating the existence of two desensitized forms of the receptor, instead of one. A second modification deals with the number of agonist molecules bound to the receptor. Biochemical studies have revealed the existence of two agonist binding sites per receptor, one on each a subunit (see, e.g. Raftery, Hunkapiller, Strader & Hood, 198). The fraction of open channels increases more than linearly with the agonist concentration, probably because a channel opens when the two binding sites are occupied (see Katz & Thesleff, 1957; Rang, 1971; Dionne, Steinbach & Stevens, 1978; Sakmann & Adams, 1978; Trautmann, 198). So, it is not surprising if more than one agonist molecule can also bind to the desensitized receptor. In fact, the 'dose-response curve for desensitization' (Fig. 3 and see Appendix) suggests that the desensitized receptors can bind two agonist molecules. APPENDIX Steady-state desensitization The cyclic model of desensitization (Katz & Thesleff, 1957) may be written: M R K AR K, D 1 AD where K1 and K1 are the dissociation constants of the agonist A, respectively for the R (activatable) and D (desensitized) forms of the receptor, and M = D/R. The ratio of the number of activatable receptors, R, over its maximum value Rmax is R _ M+1 Rmax 1 +A/KO+M( +A/IK1) Fig. 3 indicates that a large fraction of the receptors can be desensitized even when a small fraction of the receptors is activated. In this concentration range, we thus have A/Ko << 1 < A/K1. If moreover, the fraction of desensitized receptors in the absence of agonist is small, M << 1. Then, Rmax 1 A M R 'Kl1 This linear relation is not observed experimentally. Now, if the receptor can bind two agonist molecules independent sites, the model becomes: at two equivalent and K/2 2Ko M$J D /2 K1/2 AD x 2K1 A AD

14 27 A. FELTZ AND A. TRAUTMANN with the same hypothesis as above, i.e. M << 1 and A/KO < 1 < A/K1, the fraction of activatable receptors is given by R 1 V~~~rR(M) Rmax l+ma2/k12 max/r-1)i - Our experimental results are compatible with such a relation, and therefore are in favour of a model where the desensitized receptor can bind two agonist molecules. One should note that the evaluation of half of the receptors being desensitized in the prolonged presence of 2-3g/iM-CCh could be slightly underestimated if a potentiation of the e.p.c. by the perfused CCh occurred at room temperature (though we could not detect it), as well as at low temperature (Feltz & Trautmann, 198). Such a potentiation hidden by desensitization, and increasing with the agonist concentration, would not explain but could have rather masked our main result on steady state desensitization: that the fraction of desensitized receptors increases more than linearly with agonist concentration. Kinetics of desensitization If the binding of the agonist to the R form was much faster than all the other reactions, including the binding to the D form, one can write the cyclic model as following (keeping, for simplicity, the original model with only one binding site for A): R Ko AR k, 2 k2 I3 1Fk4 D k'5 AD I6k The onset and offset of desensitization will both become biphasic; the rate constants will be given by a long expression which simplifies considerably when (A) =, i.e. for recovery, whose two rate constants are given by: 1kl+k2, r k4+k6. (1) If kc kj, k2, k4, only one rate constant of recovery will appear. (2) If (1) is not true, then2 is likely to depend upon the nature of the agonist (since k6/k5 depends upon the agonist). Both possibilities are in contradiction with our results. But our results can be explained by a model including two different states of desensitization, D1 and D2, D1 corresponding to the 'low-affinity state' defined by biochemists, and D2, to the 'high affinity state'. In conclusion, a complete model should finally take into account all the possible conformations of the receptor; R (activatable), R* (activated), D1 and D2. A minimal model for activation/ desensitization would then be given by these states, and the transitions indicated with the continuous lines. (In addition, the transitions indicated by the dotted lines could also take place). A KM

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