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Nicotinic acetylcholine receptors in the neurones of autonomic ganglia
Journal of the Autonomic Nervous System, 21 (1987) 91-99
91
Elsevier JAN 00779
Review Article
Nicotinic acetylcholine receptors in the neurones of autonomic ganglia V.I. Skok Bogomoletz Institute of Physiology, Kieo (USSR)
Key words: Acetylcholine receptor; Ganglionic transmission; Ganglion-blocking drug
An autonomic ganglion is a unique object in the sense that it allows the study of both the physiology of synapses and the activity of single postsynaptic receptors. This lucky combination makes the nicotinic acetylcholine receptors (AChRs) of mammalian autonomic ganglion neurones an extremely favourable model for examining the molecular mechanisms responsible for normal receptor function and for drug-induced modulation of synaptic transmission. In this review article, some recent findings in these two important fields are briefly exposed.
Localization, structural and functional organization of AChRs It has been suggested from autoradiographic experiments performed with the use of labelled a-neurotoxins * that each neurone contains, as an average, 9.2 x 10 5 AChRs in mammalian sym-
* Although some a-toxins, e.g. a-bungarotoxin,do not block ganglionic transmission [10,33], they nevertheless bind to nicotinic AChRs of the autonomic ganglion neurones [24,34,591. Correspondence: V.I. Skok, BogomoletzInstitute of Physiology,
Kiev-24, U.S.S.R.
pathetic ganglia [24], and 3 × 10 6 AChRs in avian parasympathetic ganglia [12]. In dendrites and perikarya the highest density in AChRs is found in the postsynaptic membrane (575 receptors per /~m2), although there are also more sparsely distributed extrasynaptic receptors (35.6 per t~m2). The latter value is close to that found in extrajunctional membrane of skeletal muscle fibres, whereas the former one is 10-100 times lower than that at the motor end-plate [22,36]. The difference in the AChR density between postsynaptic and extrasynaptic neuronal membrane correlates with their different sensitivity to acetylcholine (ACh) [28,46]. The molecular organization and primary structure of the AChR protein has been studied in most detail in the electric organ of fish [30,41,42]. The molecule is an oligomeric glycoprotein of about 290,000 molecular mass and consists of 5 subunits of the stoichiometry Ot2fly~. The subunits are arranged as a rosette, with an ionic channel in the centre, together forming a mushroom-like structure that spans the cell membrane and is exposed both to the extra- and the intracellular space. The amino acid sequence of each subunit has recently been determined using recombinant DNA techniques. The portions of the AChR molecule responsible for ACh binding (the 'recognition centre') and for ion translocation function
0165-1838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (BiomedicalDivision)
92 (the ionic channel) have been tentatively identified using site-directed mutagenesis. According to recent results, the cysteins Cys 192 and Cys 193, as well as Cys 128 and Cys 142 of a-subunit have a specific role in ACh binding and signal transduction in the AChR molecule [36,37], which is supported by other data [61]. One of the possibilities is that these cysteins form disulphide bridges which are essential for maintaining the proper conformation of a-subunit. Moreover, the sequence Asp 138-Cys 142 is considered as a possible ACh-binding site. In recent work, the amino acid sequence of the A C h R a-subunit has been deduced from the eDNA clone expressing the sequences for the AChR of the rat phaeochromocytoma cell line that are pharmacologically similar to the nicotinic AChR of sympathetic ganglion neurones [6]. Although the region Asp 138-Cys 142 as well as the region near Cys 192-Cys 193 in sympathetic a-subunit are very close to those in muscle a-subunits, there are some differences in their amino acid sequences which might be responsible for the pharmacological differences between two AChR types. Another functionally important part of the AChR molecule is its ionic channel. Although it is commonly accepted that the ionic channel is formed by all 5 subunits, it remains uncertain what particular portions in each subunit line the channel [23,26,30,45]. There is a common agreement, however, that the channel-lining portions should contain charged amino residues that are able to bind the permeant ions. This requirement is fulfilled in the amphipathic segment in the proximity of aA372-388 [26,36]. Other evidence indicates that the region near serine in position 269 of 8-subunit is essential for the ionic channel functioning [30].
Responses of single neurone and of single AChR to preganglionic nerve impulse A single preganglionic impulse of threshold intensity evokes in the neurone of rabbit or rat superior cervical ganglion an excitatory postsynaptic current (EPSC) (Fig. 1C). This kind of response is usually recorded with the two-electrode voltage clamp technique (Fig. 1C). The EPSC is
characterized by mono-exponential dora5 ~lmc course (in the rat submandibular ganglion the EPSC has a bi-exponential decay time co!lrse indicating a more complex AChR channel kinetics than in sympathetic ganglia [43]). Mono-cxponential decay has also been observed in spontaneous miniature EPSCs (mEPSCs) recorded from sympathetic ganglion neurones. The quantal content of the EPSC, calculated from the ratio of evoked EPSC amplitude to amplitude of mEPSC recorded from the same cell, ranges from 4 to 243 [19], The mean decay time constant, ~'d, of EPSC or mEPS(' of rabbit sympathetic ganglion neurones at a membrane potential of 50 mV (which is close to resting membrane potential level) and at 34 37 ° ( , is about 5 ms [19]. This time constant is much longer than that of the end-plate current recorded from rat or mouse muscle fibres in similar conditions (0.3 ms) [21,29], and it is close to that found in rat parasympathetic ganglion neurones [43]. If measured at lower temperature (23 ° C) in rat sympathetic ganglia, ~d is about 13 ms [20]. An EPSC or an mEPSC is an integrated response of a great number of AChRs to a transient increase in ACh concentration that lasts no more than a few tenths of a millisecond. Therefore. the EPSC decay reflects the exponential distribution of the periods each single AChR ionic channel stays open, and ~d yields an estimate of the channel mean open time (see [2,15]). Further information on the AChR channel kinetics has been obtained from the analysis of single channel activity recorded by the patch-clamp method (Fig. 1B) [27] on rat superior cervical ganglion neurones [20]. In this case, a single AChR is continuously activated by ACh contained in the patch pipette. Single current pulses or bursts of pulses of various duration that correspond to channel single openings or to bursts of openings, appear in a random fashion (Fig. 1A). Two types of current, of small and large amplitude, can be seen, corresponding to mean channel conductances of 20 ps and of about 50 ps. The small amplitude currents occur more frequently than the large amplitude ones, and for this reason only the former are further analysed. When single-channel activity like that shown in Fig. 1A (recorded at - 110 mV membrane holding potential and at 23°C) is analysed, two mean
93
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+
20 ms
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I 10 nA
Fig. 1. Comparison of single nicotinic receptor channel activity (A, B) with the integral activity of the whole neurone nicotinic receptors (C) in rat superior cervical ganglion. A, B: the activity recorded using cell-attached patch clamp technique from single channel which is permanently activated by acetylcholine (1 '10 4 M) contained in the patch pipette; the simplified scheme of recording is shown in B. At bottom in B, the result of summation of single channel current bursts, some of which are shown at the top in B, is shown [20]. C: the EPSC recorded with two-electrode voltage clamp method (see recording scheme at the top) as evoked by single preganglionic stimulus (V.A. Derkach and A.A. Selyanko, unpublished results). The records were obtained at membrane potential - 1 1 0 mV and at 23-25°C.
channel open times and 4 mean channel closed times can be detected from the distribution of current pulse durations and interpulse intervals [20]. The two mean channel open times are 0.2 ms (%1) and 2.6 ms (%2), while the 4 mean closed times (rd-~'c4) are 0.1, 1.8, 80"0 and 1083 ms. The shortest closed times, ~'cl, are evidently the closures during the bursts, Thus, by ignoring all closures of up to 4 Tc, (this should include 98% of an exponentially distributed population of events with a mean of Td), one can estimate the mean burst duration. This procedure has yielded again two mean channel open times, of 0.3 ms (1-bl) and 8.5 m s (Tb2). The former value apparently corresponds to single openings that are separate from bursts, while the latter value is the mean burst duration. The value of %1 is statistically undistinguishable from To1; therefore, it has been assumed that the remaining open component initially found, %2, is the mean single open time within the burst. It should be noted that the value %2 is likely to be somewhat overestimated due to
the fact that the fastest interpulse intervals remain unresolved. The corrected ~'o2 value (see [20]) is 1.3 ms. This duration is similar to that found earlier in cultured neurones from the chick ciliary ganglion at resting m e m b r a n e potential level and at 30 ° C (1.08 ms [39]). It seemed interesting to compare the mean open times found by this analysis with that measured as the EPSC decay time constant, Td. The mean r d value obtained from the same animal (rat) and at the s a m e m e m b r a n e potential and temperature than in single channel experiments is 13.9 ms (Derkach, Selyanko and Skok, unpublished). This value is much closer to ~'b2 (8.5 ms) than to any other mean channel opening event. One can thus conclude that the nerve-released transmitter causes a burst of openings, rather than a single opening, in each channel. This suggestion has been tested by checking whether the sum of m a n y bursts of variable duration, taken occasionally from the same continuous record of single channel activity, can simulate the EPSC decay time course. As Fig. 1B demonstrates, this is exactly what has been observed (the somewhat longer ~'d in Fig. 1C than the Tb2 value quoted earlier is due to the fact that pooled results were used in Fig. 1C). It should be noted that the simulated EPSC decay is monoexponential (Fig. 1B) if only the bursts, without single openings, are summated. If the single openings observed in the same record are included, one additional, fast exponential component appears in the simulated EPSC decay. This fast exponential component, however, disappears after all summated data (including single openings) are adjusted so that their onsets correspond to a Gausian distribution with o = 0.5 ms, which is in agreement with the commonly observed S-shaped rising phase of the EPSC. The activity of single A C h R can be most simply described by the following reaction scheme [18]: k+l
fl
A + R,~ AR ~ AR* k
I
(1)
ot
where A is the agonist, R cupied by A, A R and A R * tor complexes with closed nels, respectively, and k+,,
is a receptor non-ocare the agonist-recepand open ionic chank 5, fl and a are the
94
rate constants. A possible interpretation of single channel activity in terms of reaction (1) is that the burst of channel openings of % duration corresponds to a series of fast transitions between A R and A R * , the inverse of the burst duration, ~-d-1, being the 'apparent' rate constant ao, while the inverse of the single opening duration, %-1, is the 'true' rate constant a of channel closing. It has been assumed that the channel closures ~c3 and %4 may correspond to desensitised states of AChRs, for their values are not much different from two kinetic components of desensitisation found by other authors in nicotinic AChRs; the remaining ~'c2 closure has been assumed to correspond to the R state in reaction 1 (for further details see [20]). On these assumptions, the following mean values for the rate constants in reaction scheme (1) have been found from distributions of channel open and channel closed times in records similar to that shown in Fig. 1A: a = 8 9 4 s - l ; f l = 6 2 9 3 s - l ; k_l = 1235 s - l ; k+l = 2.3 × 107 M 1 . s-1 [20]. These rate constants are considerably slower in the sympathetic ganglion neurone than in the motor end-plate [cf. 16,17]. Thus in sympathetic ganglion neurones both the single opening of the AChR channel and the burst of single channel openings triggered by the interaction of AChR and ACh are much longer than at the end-plate. It has been found that mean amplitude of mEPSC in sympathetic ganglion neurones is about one order of magnitude lower than in motor endplate, which correlates with a difference in the conductance change produced by a single ACh quantum (see [19]). This difference is probably due to smaller number of ionic channels opened by single ACh quantum [cf. 43], as the quantal ACh content and single AChR channel conductance do not differ much between the ganglion neurone and motor end-plate. One of the possible reasons for smaller number of open channels is that the AChR density in subsynaptic membrane is 1-2 orders lower in sympathetic neurone than in motor end-plate (see [56]). However, a much smaller difference is the effectiveness of an ACh quantum between the ganglion and neuromuscular junction can be predicted if the amount of charge transferred across the open AChR channels is compared, because of the
longer channel lifetime and mEPSC decay in the ganglion neurones. One thus can speculate that comparatively long channel lifetime in sympathetic AChRs is functionally important to compensate the small number of AChR channels opened by the ACh quantum. The apparent mean channel open time in sympathetic neuronal AChRs can be markedly increased by raising the external calcium concentration [50]. Similar increase has been observed in the AChRs of parasympathetic neurons and of molluscan neurones [35,43], but not in the end-plate AChRs [8,31]. This difference correlates with the differences between the apparent mean channel open times, thus implying that calcium ions may stabilise the channel in its open conformation.
The AChR blocking mechanisms Recent electrophysiologicat studies have revealed that there are at least two mechanisms underlying the blockade of ganglionic AChRs by ganglion-blocking agents: blockade of open-channel. a kind of non-competitive blockade, and blockade of a recognition centre of the AChR (a competitive blockade). The open channel blockade has been widely observed in the AChRs of autonomic ganglion neurones [4,5,48] and skeletal muscle fibres [1,14]. This type of blockade is characterised by a voltage-dependent decrease in mean channel-open time, with no changes in the probability of the channel to be opened by nerve-released ACh. The latter characteristic results in that the EPSC amplitude is almost unchanged by a blocker, except for a small reduction due to channel-open time shortening, while the time constant of the EPSC decay appears markedly shortened. This is in sharp contrast with what is observed in the AChR recognition centre blockade, when the EPSC amplitude markedly decreases without a decrease in the EPSC decay time constant. The open channel blockade can be most simply described by the following scheme [1]: k+B[B]
AR* + B
~ k
AR*B, B
(2)
95
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Fig. 2. Blocking effects produced in the neurones of rabbit (Ai,2; B1 ) and rat (A 3; B2.3) superior cervical ganglion by hexamethonium ( 1 . 1 0 -5 M in A l and 5 . 1 0 -5 M in A2,3) and trimethaphan (1-10 -5 M in B 1 and 3 . 1 0 -6 M in 132.3). A a and BI: the EPSC recorded before (a) and during (b) the application of a blocking agent; membrane potential is - 5 0 mV in A and - 1 1 0 mV in B ([52]; A.Y. Bobrishev, unpublished results). A 2 and B2: membrane currents (ACh currents) evoked by paired iontophoretic applications of acetylcholine ([55]; V.A. Derkach and A.A. Selyanko, unpublished results). A 3 and B3: voltage dependence of the blocker-induced suppression of ACh currents. Abscissa, membrane potential; ordinate, relative suppression of control ACh current I 0 to I level. Some I o and I currents recorded at the same membrane potential without a blocker and in the presence of a blocker are superimposed in the inset. The blocking effect is voltage-dependent in A and voltage-independent in B (V.A. Derkach and A.A. Selyanko, unpublished results).
where A R * is the agonist-receptor complex with open channel, B is a blocking agent in concentration [B], A R * B is the open-blocked form, and k+B and k B are the rate constants. The open channel blockade is indicated, in particular, by a voltage-dependent decrease in Td without a decrease, at least at threshold concentrations of the blocking agent, of the EPSC amplitude (Fig. 2A1), and by frequency-dependent (Fig. 2A 2) and voltage-dependent (Fig. 2A 3) decrease in the ACh-induced current. This effect has been produced in mammalian sympathetic and parasympathetic ganglion neurones by hexamethonium (Fig. 2A), by other bis-quaternary ammonium compounds, by pirilenum, a tertiary amine, and by other
specific ganglion-blocking agents [4,25,48,49,53, 55,56]. The results are consistent with the recent observation that hexamethonium (1 × 10 -5 M) does not produce a parallel shift in dose-response curves obtained basing upon the changes of the ACh-induced current in rat superior cervical ganglion neurones (V.A. Derkach and A.S. Selyanko, unpublished observations). It follows from equations (1) and (2) and from the above description of single-channel activity that the mean 'true' channel-open time estimated in the presence of a blocking agent, "rff,is given by •o =
+ k+
[Bl]
(3)
96
Because the EPSC decay time constant, ~'d, becomes markedly shortened in presence of the AChR open-channel blocker (Fig. 2A1), one should expect that mean burst time, %, would likewise be shortened. This conclusion is what follows from theoretical predictions of the effects produced by open-channel blockers [15] if instead of fast-dissociating blockers, e.g. local anaesthetics, a slow-dissociating blocker, e.g. hexa- or heptamethonium, is used (for more detailed discussion on this point see [56]). The results obtained with heptamethonium on single AChR channel activity in rat superior cervical ganglion neurones [20] have confirmed this conclusion. Both r b and %2 are shortened by heptamethonium, the former much more than the latter. The 'apparent' k+ B value, k '+B, as estimated from an equation similar to (3) but with % and r£ (burst duration in the presence of a blocker) instead of % and %', yields a mean k '+ B value of 18 x 10 6 M - ] . s -1, while the mean k+~ value is 65 × 106 M -1 . s -I
[201. An easier way to obtain k~B is to replace in equation (3) % and %' with the EPSC decay time constants measured in normal ( r d) and in blocker-containing (~-d) solutions. The k'_ B value can be estimated as the inverse of the time constant that characterises the restoration of AChinduced current evoked at various intervals after the conditional ACh application (Fig, 2A 2)- From k+B and k ' B, the affinity constant K B' = k+' 8 / k '- B can be found. Fig. 2A 3 shows that there is no voltage-independent component in the blocking effect produced by hexamethonium on the ACh-induced current, as the blocking effect drops to zero at positive membrane potential levels. On the other hand, it was found both in parasympathetic [4] and in sympathetic [48] ganglia that the k '+ B value for hexamethonium is higher than that of tubocurarine. Because hexamethonium is a specific ganglionic blocker, in contrast to tubocurarine, it was suggested that this difference is due to the open-channel-blocking effect which in this case determines the selectivity of ganglionic blockade [48]. To check this suggestion, the k'+e and Ke values were determined for several specific ganglion-blocking agents and were compared with
]
;Ai[(;LION-~ILOCK!N; A~:'~[V2'
[ ] k'+B [] K'B
% 1000 1/30
10
O.I
O. Ol
IEH
C4
C5
C6
C,,.;.
P
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Fig. 3. Comparison of ganglion-blocking activity with channel-blocking activity for ganglion-blocking drugs IEM1119, tetra-(C4), penta-(Cs), hexa-(Cr), hepta.methonium (C7) and pirilenum (P) as indicated at the abscissa [53,55]i
their potencies to block ganglionic transmission [55] (Fig. 3). The ganglion-blocking potency of all the agents tested correlates ( P > 0.95) with their t t k+B values (but not with their K B values). Therefore, the rate of binding of a blocker to an open channel is most important in the blockade of synaptic transmission [53 56]. The lack of correlation between the ganglion-blocking potencies and K~ values is a result of two factors; (1) the k values differ among the drugs used much less than the k~B values, and (2) the frequencms of the preganglionic stimuli applied to test transmission blockade are too low to detect significant differences in k'_ s values between the drugs used. A similar correlation between the open-channel-blocking and ganglion-blocking activities was later observed by Gurney and Rang [25] with the short-chain bis-quaternary ammonium compounds mentioned above, but not with long-chain bisquaternary ammonium compounds (longer than C7), which are not specific gan$1ion blockers. Most recent experiments revealed a new class of very specific blockers for synaptic transmission through enteric nervous plexuses which are char-
97 acterised by extremely long action [56]. The investigation of mechanisms of their action suggests that they combine the open-channel blocking effect with irreversible binding to the A C h R molecule (Gmiro, V.E., Derkach, V.A., Kurrenny, D.E. et al., in press). The second mechanism, the blockade of a recognition centre, is illustrated by the effect produced with trimethaphan, a specific ganglionblocking agent. Trimethaphan, unlike hexamethonium, decreases the amplitude of EPSC in sympathetic neurones without decrease in % (Fig. 2 B1) [7]. Another marked difference from hexamethonium is that the effect of trimethaphan on the ACh-induced current is frequency-independent (Fig. 2B2) and voltage-independent (Fig. 2B3); the membrane hyperpolarization is not followed by an increase in the trimethaphan-induced blockade. Thus, trimethaphan does not produce open-channel blockade. These results are consistent with earlier observations on the rat submandibular ganglion where trimethaphan is a pure competitive blocking agent [4]. Pure competitive effects on ganglionic AChRs have also been observed with a-neurotoxins, in particular, with surugatoxin [see 9], a-bungarotoxin and acobratoxin [47]. It can be concluded that specific ganglionic blockade produced by different drugs is due to a blockade of either the A C h R open channel or the AChR recognition center. The blockade of synaptic transmission through the blockade of the open postsynaptic channels, which may at first look surprising, is due to a decrease in the electric charge carried by the EPSC, which in turn causes a decrease in the amplitude of postsynaptic potential, thus making it subthreshold for the postsynaptic spike initiation [53,54]. The drugs which produce the specific openchannel blockade in nicotinic AChRs of symphathetic ganglion neurones, do this also in nicotinic AChRs of the neurones of parasympathetic ganglia and of molluscan neurones [3], but not in those of motor end-plates [32,44]. This difference correlates with the sensitivity of the A C h R channels to calcium ions that can prolong mean channel-open time (see above). One may suggest that the specific open channel-blocking drugs bind to
the sites in the channel that normally interact with calcium ions. This suggestion is consistent with the observation that increased external concentration of calcium ions strongly inhibits binding of hexamethonium to the open channel, as indicated by the marked drop in k '+8 in sympathetic ganglion neurones [50]. Moreover, calcium-binding sites have been found in the ionic channels of nicotinic AChRs (in molluscan neurones [11,57]). To summarise the characteristics of nicotinic acetylcholine receptors in autonomic ganglion neurones, one should emphasise that they are unique in at least 3 respects, when compared with nicotinic acetylcholine receptors in other tissues: (1) in the long mean channel-open time, both 'true' and 'apparent', which determines long-lasting postsynaptic current decay; (2) in the high sensitivity of the channel kinetics to increased concentration of calcium ions; (3) in the susceptibility of the open channel to drug-induced blockade, which underlies the specificity of many ganglionic blockers. It seems likely that all 3 characteristics relate to the same peculiarity of the channel-operating mechanism which possibly involves calcium ions.
References 1 Adams, P.R., Drug blockade of open end-plate channels, J. Physiol. (London), 260 (1976) 531-552. 2 Anderson, C.R. and Stevens, C.F., Voltage clamp analysis of acetylcholine-produced end-plate current fluctuations at frog neuromuscular junction, J. Physiol. (London), 235 (1973) 655-691. 3 Ascher, P., Marry, A. and Neild, T.O., The mode of action of antagonists on the excitatory response to acetylcholine in Aplysianeurones, J. Physiol. (London), 278 (1978) 207-235. 4 Ascher, P., Large, W.A. and Rang, H., Studies on the mechanism of action of acetylcholine antagonists on rat parasympathetic ganglion cells. J. Physiol. (London), 295 (1979) 139-170. 5 Blackman, S.J., Dependence on membrane potential of the blocking action of hexamethonium at a sympathetic ganglionic synapse, Proc. Univ. Otago Med. Sch., 48 (1970) 4-5. 6 Boulter, J., Evans, K., Goldman, D. et al., Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor a-subunit, Nature (London), 319 (1986) 368-174.
98 7 Bobrishev, A.Y. and Skok, V.I., Action of trimethaphan on excitatory postsynaptic current in nerve cell of sympathetic ganglion, Proc. Ukr. SSR Acad. Sci., 5 (1987) 60-61. 8 Bregestovsky, P.D., Miledi, R. and Parker, 1., Calcium conductance of acetylcholine-induced endplate channels, Nature (London), 279 (1979) 638-639. 9 Brown, D.A., Locus and mechanism of action of ganglionblocking agents. In D.A. Kharkevich (Ed.), Pharmacology of Ganglionic Transmission, Springer, Berlin, 1980, pp. 185-234. 10 Brown, D.A. and Fumagalli, L., Dissociation of abungarotoxin binding and receptor block in the rat superior cervical ganglion, Brain Res., 129 (1977) 165-168. 11 Chemeris, N.K., Kazachenko, V.N., Kislov, A.N. and Kurchikov, A.L., Inhibition of acetylcholine responses by intracellular calcium in Limnea stagnalis neurones, J. Physiol. (London), 323 (1982) 1-9. 12 Chiappinelli, V.A. and Giacobini, E., Time course of appearance of a-binding sites during development of chick ciliary ganglion and iris, Neurochem. Res., 3 (1978) 465-478. 13 Clarke, P.B.S., Schwartz, R.D., Paul, S.M. et al., Nicotinic binding in rat brain: autoradiographic comparison of 3Hacetylcholine, 3H-nicotine, and 125I-a-bungarotoxin, J. Neurosci., 5 (1985) 1307-1315. 14 Colquhoun, D., Dreyer, F. and Sheridan, R.E., The actions of tubocurarine at the frog neuromuscular junction, J. Physiol. (London), 293 (1979) 247-284. 15 Colquhoun, D. and Hawkes, A.G., The principles of the stochastic interpretation of ion-channel mechanisms, in E. Neher and B. Sakmann (Eds.), Single-Channel Recording, Plenum, New York, 1983, pp. 135-174. 16 Colquhoun, D. and Sakmann, B., Bursts of openings in transmitter-activated ion channels, In E. Neher and B. Sakmann, (Eds.), Single-Channel Recording, Plenum, New York, 1983, pp. 345-364. 17 Connor, E.A., Neel, D.S. and Parsons, R.L., Influence of the extracellular ionic environment on ganglionic fast excitatory postsynaptic currents, Brain Res., 339 (1985) 227-235. 18 Del Castillo, J. and Katz, B., Interaction at end-plate receptors between different choline derivatives, Proc. R. Soc. London Ser. B., 146 (1957) 369-381. 19 Derkach, V.A., Selyanko, A.A. and Skok, V.I., Acetylcholine-induced current fluctuations and fast excitatory postsynaptic currents in rabbit sympathetic neurones, J. Physiol. (London), 336 (1983) 511-526. 20 Derkach, V.A., North, R.A., Selyanko, A.A. and Skok, V.I., Single channels activated by aeetylcholine in rat superior cervical ganglion, J. Physiol. (London) 388 (1987) 141-151. 21 Dreyer, F., Miiller, K.D., Peper, K. and Sterz, R., The M. omohyoideus of the mouse as a convenient mammalian muscle prepatation, Pfliigers Arch., 367 (1976) 115-122. 22 Fertuck, N.C. and Salpeter, M.M., Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 12~I-a-bungarotoxin binding at mouse neuromuscular junctions, J. Cell. Biol., 69 (1976) 144-158.
23 Finer-Moore, J. and Stroud, R.M., Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor, Proc. Natl. Acad. Sci. U.S.A.. 81 (1984) 155-159. 24 Fumagalli, L., De Renzis, G. and Miani, N., Acetylcho!ine receptors: number and distribution in intact and deafferented superior cervical ganglion of the rat, J Neurochem.. 27 (1976) 47-52. 25 Gurney, A.M. and Rang, H.P., The channel-blocking action of methonium compounds on rat submandibular ganglion cells, Br. J. PharmacoL, 82 (1984) 623-642. 26 Guy, H.R., A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations, Biophys. J., 45 (1984) 249-261. 27 Hamill, O.P., Marty, A., Neher, E. et al., Improved patchclamp techniques for high-resohition current recording from cells and cell-free membrane patches, Pfliiger~ Arch., 391 (1981) 85-100. 28 Harris, A.J., Kuffler, S.W. and Dennis, M.J., Differential chemosensitivity of synaptic and extrasynaptic areas on the neuronal surface membrane in parasympathetic neurons on the frog tested by micro-application of acetylcholine, Proc R. Soc. London Ser. B., 177 (1971) 541-553. 29 Head, S.D., Temperature and end-plate currents in rat diaphragm, J. Physiol. (London), 334 (1983) 441-459. 30 Hucho, F., The nicotine acetylcholine receptor ion channel, Eur J. Biochem., 158 (1986) 211-226. 31 Kuba, K. and Takeshita, S., On the mechanism of calcium action on the acetylcholine receptor-channel complex at the frog end-plate membrane. Jpn. J. Physiol.. 33 (1983) 931-944. 32 Lukomskaya, N.Ya., Magazanik, L.G. and Potapieva, N.N., Effect of bis-onium gangliolytics on kinetics of synaptic ionic channels activation, In V.I. Skok (Ed.), Physiology of Autonomic Ganglia, Naukova, Dumka, Kiev, 1981; pp. 62-63. 33 Magazanik, L.G., tvanov, A.Ya. and Lukomskaya, N.Y., The effect of snake venom polypeptides o n cholinoreceptors in isolated rabbit ganglia, Nuerofiziol., 6 (1974) 652-656. 34 Marshall, L.M., Synaptic localization of a-bungarotoxin binding which blocks nicotinic transmission at frog symphathetic neurons, Proc. Natl. Acad Sci. U.S,A., 78 (1981) 1948-1952. 35 Marty, A., Action of calcium ions on acetylcholine-sensitive channels in Aplysia neurones, J. Physiol. (Paris), 76 (1980) 523-527. 36 Mishina, M., Tobimatsu, T., Imoto, K. et al., Location of functional regions of acetylcholine receptor a-subunit by site-directed mutagenesis, Nature (London), 313 (1985) 364-369. 37 Noda, M., Takahashi, H., Tanabe, T. et al., Primary structure of a-subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence, Nature (London), 299 (1982) 793-797. 38 Noda, M., Furutani, Y., Takahashi, H. et al., Cloning and sequence analysis of calf cDNA and human genomic DNA
99
39
40
41
42
43
44
45
46
47
48
49
encoding a-subunit precursor of molecular acetylcholine receptor, Nature (London), 305 (1983) 818-823. Ogden, D.C., Gray, P.T.A., Colquhoun, D. and Rang, H.P., Kinetics of acetylcholine activated ion channels in chick ciliary ganglion neurones grown in tissue culture, Pfliigers Arch., 400 (1984) 44-50. Paton, W.D.M. and Zaimis, E.J., The pharmacological action of polymethylene bistrimethyl ammonium salts, Br. J. Pharmacol., 4 (1949) 381-400. Popot, J. and Changeux, J., Nicotinic receptor of acetylcholine: structure of an oligomeric integral membrane protein, PhysioL Rev., 64 (1984) 1162-1239. Raftery, M.A., Dunn, S.M.J., Conti-Tronconi, B.M. et al., The nicotinic acetylcholine receptor: subunit structure, functional binding sites, and ion transport properties, Cold Spring Harbor Symp. Quant. Biol., 48 (1983) 21-34. Rang, H.P., The characteristics of synaptic currents and responses to acetylcholine of rat submandibular ganglion cells, J. Physiol. (London), 311 (1981) 23-55. Rang, H.P. and Rylett, R.J., The interaction between hexamethonium and tubocurarine on the rat neuromuscular junction, Br. J. Pharmacol., 84 (1984) 519-531. Ratnam, M., Le Nguyen, D., Rivier, J. et al., Transmembrane topography of nicotine acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles, Biochemistry, 25 (1986) 2633-2643. Roper, S., The acetylcholine sensitivity of the surface membrane of multiply-innervated parasympathetic ganglion cells in the mudpuppy before and after partial denervation, J. Physiol. (London), 254 (1976) 455-473. Selyanko, A.A., Mechanism of the action of a-neurotoxins form the snake venoms on nicotinic acetylcholine receptors in rabbit sympathetic ganglion neurons, Neurofiziol., 15 (1983) 377-382. Selyanko, A.A., Derkach, V.A. and Skok, V.I., Effects of some ganglion-blocking agents on fast excitatory postsynaptic currents in mammalian sympathetic ganglion neurones. In J. Salanki (Ed.), Physiology of Excitable Membranes, Akad. Kiado, Budapest, 4, 1981, pp. 329-342. Selyanko, A.A., Derkach, V.A. and Skok, V.I., Voltagedependent actions of short-chain polymethylene bis-trimethylammonium compounds on sympathetic ganglion neurons, J. Auton. Nert,. Syst., 6 (1982) 13-21.
50 Selyanko, A.A., Derkach, V.A. and Skok, V.I., The effect of Ca2+-ions on the channel-blocking action of hexamethonium in sympathetic ganglion, Proc. USSR Acad. Sci, 284 (1985) 225-228. 51 Shibahara, S., Kubo, T., Perski, H.J. et al, Cloning and sequence analysis of human genomic DNA encoding subunit precursor of muscle acetylcholine receptor, Eur. J. Biochem., 146 (1985) 15-22. 52 Skok, V.I., Selyanko, A.A., Derkach, V.A., Gmiro, V.E. and Lukomskaya, N.Y., The mechanisms of ganglion-blocking action of bis-ammonium compounds, Neurofiziol., 16 (1984) 54-60. 53 Skok, V.I., Channel-blocking mechanism ensures specific blockade of synaptic transmission, Neuroscience, 17 (1986) 1-9. 54 Skok, V.I., The blockade of open channel of acetylcholine receptor is responsible for selective blockade of nicotinic transmission, In N.J. Dun and R.L. Perlman (Eds.), Neurobiology of Acetylcholine, Plenum, New York, 1987. 55 Skok, V.I., Selyanko, A.A. and Derkach, V.A, Channelblocking activity is a possible mechanism for a selective ganglionic blockade, Pfliigers Arch., 398 (1983) 169-171. 56 Skok, V.I., Selyanko, A.A. and Derkach, V.A., Neuronal Acetylcholine Receptors, Nauka, Moskow, 1987. 57 Slater, N.T., Carpenter, D.O, Haas, H.L and David, J.A., Blocking kinetics at excitatory acetylcholine responses on Aplysia neurons, Biophys. J., 45 (1984) 24-25. 58 Smart, L., Meyers, H. and Hilgenfeld, R., A structural model for the ligand-binding sites at the nicotinic acetylcholine receptor, FEBS Lett., 178 (1984) 64-68. 59 Smolen, A.J., Specific binding of a-bungarotoxin to synaptic membranes in rat sympathetic ganglion:computer best-fit analysis of electron microscope radioautographs, Brain Res., 289 (1983) 177-188. 60 Tanabe, T., Noda, M., Furutani, Y. et al., Primary structure of/3-subunit precursor of calf muscle acetylcholine receptor deduced from cDNA sequence, Eur. J. Biochem., 144 (1984) 11-17. 61 Wilson, P.T., Lentz, T.L. and Hawrot, E., Determination of the primary amino acid sequence specifiying the abungarotoxin binding site on the a subunit of the acetylcholine receptor from Torpedo californica, Neurobiology, 82 (1985) 8790-8794.
91
Elsevier JAN 00779
Review Article
Nicotinic acetylcholine receptors in the neurones of autonomic ganglia V.I. Skok Bogomoletz Institute of Physiology, Kieo (USSR)
Key words: Acetylcholine receptor; Ganglionic transmission; Ganglion-blocking drug
An autonomic ganglion is a unique object in the sense that it allows the study of both the physiology of synapses and the activity of single postsynaptic receptors. This lucky combination makes the nicotinic acetylcholine receptors (AChRs) of mammalian autonomic ganglion neurones an extremely favourable model for examining the molecular mechanisms responsible for normal receptor function and for drug-induced modulation of synaptic transmission. In this review article, some recent findings in these two important fields are briefly exposed.
Localization, structural and functional organization of AChRs It has been suggested from autoradiographic experiments performed with the use of labelled a-neurotoxins * that each neurone contains, as an average, 9.2 x 10 5 AChRs in mammalian sym-
* Although some a-toxins, e.g. a-bungarotoxin,do not block ganglionic transmission [10,33], they nevertheless bind to nicotinic AChRs of the autonomic ganglion neurones [24,34,591. Correspondence: V.I. Skok, BogomoletzInstitute of Physiology,
Kiev-24, U.S.S.R.
pathetic ganglia [24], and 3 × 10 6 AChRs in avian parasympathetic ganglia [12]. In dendrites and perikarya the highest density in AChRs is found in the postsynaptic membrane (575 receptors per /~m2), although there are also more sparsely distributed extrasynaptic receptors (35.6 per t~m2). The latter value is close to that found in extrajunctional membrane of skeletal muscle fibres, whereas the former one is 10-100 times lower than that at the motor end-plate [22,36]. The difference in the AChR density between postsynaptic and extrasynaptic neuronal membrane correlates with their different sensitivity to acetylcholine (ACh) [28,46]. The molecular organization and primary structure of the AChR protein has been studied in most detail in the electric organ of fish [30,41,42]. The molecule is an oligomeric glycoprotein of about 290,000 molecular mass and consists of 5 subunits of the stoichiometry Ot2fly~. The subunits are arranged as a rosette, with an ionic channel in the centre, together forming a mushroom-like structure that spans the cell membrane and is exposed both to the extra- and the intracellular space. The amino acid sequence of each subunit has recently been determined using recombinant DNA techniques. The portions of the AChR molecule responsible for ACh binding (the 'recognition centre') and for ion translocation function
0165-1838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (BiomedicalDivision)
92 (the ionic channel) have been tentatively identified using site-directed mutagenesis. According to recent results, the cysteins Cys 192 and Cys 193, as well as Cys 128 and Cys 142 of a-subunit have a specific role in ACh binding and signal transduction in the AChR molecule [36,37], which is supported by other data [61]. One of the possibilities is that these cysteins form disulphide bridges which are essential for maintaining the proper conformation of a-subunit. Moreover, the sequence Asp 138-Cys 142 is considered as a possible ACh-binding site. In recent work, the amino acid sequence of the A C h R a-subunit has been deduced from the eDNA clone expressing the sequences for the AChR of the rat phaeochromocytoma cell line that are pharmacologically similar to the nicotinic AChR of sympathetic ganglion neurones [6]. Although the region Asp 138-Cys 142 as well as the region near Cys 192-Cys 193 in sympathetic a-subunit are very close to those in muscle a-subunits, there are some differences in their amino acid sequences which might be responsible for the pharmacological differences between two AChR types. Another functionally important part of the AChR molecule is its ionic channel. Although it is commonly accepted that the ionic channel is formed by all 5 subunits, it remains uncertain what particular portions in each subunit line the channel [23,26,30,45]. There is a common agreement, however, that the channel-lining portions should contain charged amino residues that are able to bind the permeant ions. This requirement is fulfilled in the amphipathic segment in the proximity of aA372-388 [26,36]. Other evidence indicates that the region near serine in position 269 of 8-subunit is essential for the ionic channel functioning [30].
Responses of single neurone and of single AChR to preganglionic nerve impulse A single preganglionic impulse of threshold intensity evokes in the neurone of rabbit or rat superior cervical ganglion an excitatory postsynaptic current (EPSC) (Fig. 1C). This kind of response is usually recorded with the two-electrode voltage clamp technique (Fig. 1C). The EPSC is
characterized by mono-exponential dora5 ~lmc course (in the rat submandibular ganglion the EPSC has a bi-exponential decay time co!lrse indicating a more complex AChR channel kinetics than in sympathetic ganglia [43]). Mono-cxponential decay has also been observed in spontaneous miniature EPSCs (mEPSCs) recorded from sympathetic ganglion neurones. The quantal content of the EPSC, calculated from the ratio of evoked EPSC amplitude to amplitude of mEPSC recorded from the same cell, ranges from 4 to 243 [19], The mean decay time constant, ~'d, of EPSC or mEPS(' of rabbit sympathetic ganglion neurones at a membrane potential of 50 mV (which is close to resting membrane potential level) and at 34 37 ° ( , is about 5 ms [19]. This time constant is much longer than that of the end-plate current recorded from rat or mouse muscle fibres in similar conditions (0.3 ms) [21,29], and it is close to that found in rat parasympathetic ganglion neurones [43]. If measured at lower temperature (23 ° C) in rat sympathetic ganglia, ~d is about 13 ms [20]. An EPSC or an mEPSC is an integrated response of a great number of AChRs to a transient increase in ACh concentration that lasts no more than a few tenths of a millisecond. Therefore. the EPSC decay reflects the exponential distribution of the periods each single AChR ionic channel stays open, and ~d yields an estimate of the channel mean open time (see [2,15]). Further information on the AChR channel kinetics has been obtained from the analysis of single channel activity recorded by the patch-clamp method (Fig. 1B) [27] on rat superior cervical ganglion neurones [20]. In this case, a single AChR is continuously activated by ACh contained in the patch pipette. Single current pulses or bursts of pulses of various duration that correspond to channel single openings or to bursts of openings, appear in a random fashion (Fig. 1A). Two types of current, of small and large amplitude, can be seen, corresponding to mean channel conductances of 20 ps and of about 50 ps. The small amplitude currents occur more frequently than the large amplitude ones, and for this reason only the former are further analysed. When single-channel activity like that shown in Fig. 1A (recorded at - 110 mV membrane holding potential and at 23°C) is analysed, two mean
93
B 13pA
q2"
0
"~
+
20 ms
2D - rns
I 10 nA
Fig. 1. Comparison of single nicotinic receptor channel activity (A, B) with the integral activity of the whole neurone nicotinic receptors (C) in rat superior cervical ganglion. A, B: the activity recorded using cell-attached patch clamp technique from single channel which is permanently activated by acetylcholine (1 '10 4 M) contained in the patch pipette; the simplified scheme of recording is shown in B. At bottom in B, the result of summation of single channel current bursts, some of which are shown at the top in B, is shown [20]. C: the EPSC recorded with two-electrode voltage clamp method (see recording scheme at the top) as evoked by single preganglionic stimulus (V.A. Derkach and A.A. Selyanko, unpublished results). The records were obtained at membrane potential - 1 1 0 mV and at 23-25°C.
channel open times and 4 mean channel closed times can be detected from the distribution of current pulse durations and interpulse intervals [20]. The two mean channel open times are 0.2 ms (%1) and 2.6 ms (%2), while the 4 mean closed times (rd-~'c4) are 0.1, 1.8, 80"0 and 1083 ms. The shortest closed times, ~'cl, are evidently the closures during the bursts, Thus, by ignoring all closures of up to 4 Tc, (this should include 98% of an exponentially distributed population of events with a mean of Td), one can estimate the mean burst duration. This procedure has yielded again two mean channel open times, of 0.3 ms (1-bl) and 8.5 m s (Tb2). The former value apparently corresponds to single openings that are separate from bursts, while the latter value is the mean burst duration. The value of %1 is statistically undistinguishable from To1; therefore, it has been assumed that the remaining open component initially found, %2, is the mean single open time within the burst. It should be noted that the value %2 is likely to be somewhat overestimated due to
the fact that the fastest interpulse intervals remain unresolved. The corrected ~'o2 value (see [20]) is 1.3 ms. This duration is similar to that found earlier in cultured neurones from the chick ciliary ganglion at resting m e m b r a n e potential level and at 30 ° C (1.08 ms [39]). It seemed interesting to compare the mean open times found by this analysis with that measured as the EPSC decay time constant, Td. The mean r d value obtained from the same animal (rat) and at the s a m e m e m b r a n e potential and temperature than in single channel experiments is 13.9 ms (Derkach, Selyanko and Skok, unpublished). This value is much closer to ~'b2 (8.5 ms) than to any other mean channel opening event. One can thus conclude that the nerve-released transmitter causes a burst of openings, rather than a single opening, in each channel. This suggestion has been tested by checking whether the sum of m a n y bursts of variable duration, taken occasionally from the same continuous record of single channel activity, can simulate the EPSC decay time course. As Fig. 1B demonstrates, this is exactly what has been observed (the somewhat longer ~'d in Fig. 1C than the Tb2 value quoted earlier is due to the fact that pooled results were used in Fig. 1C). It should be noted that the simulated EPSC decay is monoexponential (Fig. 1B) if only the bursts, without single openings, are summated. If the single openings observed in the same record are included, one additional, fast exponential component appears in the simulated EPSC decay. This fast exponential component, however, disappears after all summated data (including single openings) are adjusted so that their onsets correspond to a Gausian distribution with o = 0.5 ms, which is in agreement with the commonly observed S-shaped rising phase of the EPSC. The activity of single A C h R can be most simply described by the following reaction scheme [18]: k+l
fl
A + R,~ AR ~ AR* k
I
(1)
ot
where A is the agonist, R cupied by A, A R and A R * tor complexes with closed nels, respectively, and k+,,
is a receptor non-ocare the agonist-recepand open ionic chank 5, fl and a are the
94
rate constants. A possible interpretation of single channel activity in terms of reaction (1) is that the burst of channel openings of % duration corresponds to a series of fast transitions between A R and A R * , the inverse of the burst duration, ~-d-1, being the 'apparent' rate constant ao, while the inverse of the single opening duration, %-1, is the 'true' rate constant a of channel closing. It has been assumed that the channel closures ~c3 and %4 may correspond to desensitised states of AChRs, for their values are not much different from two kinetic components of desensitisation found by other authors in nicotinic AChRs; the remaining ~'c2 closure has been assumed to correspond to the R state in reaction 1 (for further details see [20]). On these assumptions, the following mean values for the rate constants in reaction scheme (1) have been found from distributions of channel open and channel closed times in records similar to that shown in Fig. 1A: a = 8 9 4 s - l ; f l = 6 2 9 3 s - l ; k_l = 1235 s - l ; k+l = 2.3 × 107 M 1 . s-1 [20]. These rate constants are considerably slower in the sympathetic ganglion neurone than in the motor end-plate [cf. 16,17]. Thus in sympathetic ganglion neurones both the single opening of the AChR channel and the burst of single channel openings triggered by the interaction of AChR and ACh are much longer than at the end-plate. It has been found that mean amplitude of mEPSC in sympathetic ganglion neurones is about one order of magnitude lower than in motor endplate, which correlates with a difference in the conductance change produced by a single ACh quantum (see [19]). This difference is probably due to smaller number of ionic channels opened by single ACh quantum [cf. 43], as the quantal ACh content and single AChR channel conductance do not differ much between the ganglion neurone and motor end-plate. One of the possible reasons for smaller number of open channels is that the AChR density in subsynaptic membrane is 1-2 orders lower in sympathetic neurone than in motor end-plate (see [56]). However, a much smaller difference is the effectiveness of an ACh quantum between the ganglion and neuromuscular junction can be predicted if the amount of charge transferred across the open AChR channels is compared, because of the
longer channel lifetime and mEPSC decay in the ganglion neurones. One thus can speculate that comparatively long channel lifetime in sympathetic AChRs is functionally important to compensate the small number of AChR channels opened by the ACh quantum. The apparent mean channel open time in sympathetic neuronal AChRs can be markedly increased by raising the external calcium concentration [50]. Similar increase has been observed in the AChRs of parasympathetic neurons and of molluscan neurones [35,43], but not in the end-plate AChRs [8,31]. This difference correlates with the differences between the apparent mean channel open times, thus implying that calcium ions may stabilise the channel in its open conformation.
The AChR blocking mechanisms Recent electrophysiologicat studies have revealed that there are at least two mechanisms underlying the blockade of ganglionic AChRs by ganglion-blocking agents: blockade of open-channel. a kind of non-competitive blockade, and blockade of a recognition centre of the AChR (a competitive blockade). The open channel blockade has been widely observed in the AChRs of autonomic ganglion neurones [4,5,48] and skeletal muscle fibres [1,14]. This type of blockade is characterised by a voltage-dependent decrease in mean channel-open time, with no changes in the probability of the channel to be opened by nerve-released ACh. The latter characteristic results in that the EPSC amplitude is almost unchanged by a blocker, except for a small reduction due to channel-open time shortening, while the time constant of the EPSC decay appears markedly shortened. This is in sharp contrast with what is observed in the AChR recognition centre blockade, when the EPSC amplitude markedly decreases without a decrease in the EPSC decay time constant. The open channel blockade can be most simply described by the following scheme [1]: k+B[B]
AR* + B
~ k
AR*B, B
(2)
95
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~ 5 n A
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Fig. 2. Blocking effects produced in the neurones of rabbit (Ai,2; B1 ) and rat (A 3; B2.3) superior cervical ganglion by hexamethonium ( 1 . 1 0 -5 M in A l and 5 . 1 0 -5 M in A2,3) and trimethaphan (1-10 -5 M in B 1 and 3 . 1 0 -6 M in 132.3). A a and BI: the EPSC recorded before (a) and during (b) the application of a blocking agent; membrane potential is - 5 0 mV in A and - 1 1 0 mV in B ([52]; A.Y. Bobrishev, unpublished results). A 2 and B2: membrane currents (ACh currents) evoked by paired iontophoretic applications of acetylcholine ([55]; V.A. Derkach and A.A. Selyanko, unpublished results). A 3 and B3: voltage dependence of the blocker-induced suppression of ACh currents. Abscissa, membrane potential; ordinate, relative suppression of control ACh current I 0 to I level. Some I o and I currents recorded at the same membrane potential without a blocker and in the presence of a blocker are superimposed in the inset. The blocking effect is voltage-dependent in A and voltage-independent in B (V.A. Derkach and A.A. Selyanko, unpublished results).
where A R * is the agonist-receptor complex with open channel, B is a blocking agent in concentration [B], A R * B is the open-blocked form, and k+B and k B are the rate constants. The open channel blockade is indicated, in particular, by a voltage-dependent decrease in Td without a decrease, at least at threshold concentrations of the blocking agent, of the EPSC amplitude (Fig. 2A1), and by frequency-dependent (Fig. 2A 2) and voltage-dependent (Fig. 2A 3) decrease in the ACh-induced current. This effect has been produced in mammalian sympathetic and parasympathetic ganglion neurones by hexamethonium (Fig. 2A), by other bis-quaternary ammonium compounds, by pirilenum, a tertiary amine, and by other
specific ganglion-blocking agents [4,25,48,49,53, 55,56]. The results are consistent with the recent observation that hexamethonium (1 × 10 -5 M) does not produce a parallel shift in dose-response curves obtained basing upon the changes of the ACh-induced current in rat superior cervical ganglion neurones (V.A. Derkach and A.S. Selyanko, unpublished observations). It follows from equations (1) and (2) and from the above description of single-channel activity that the mean 'true' channel-open time estimated in the presence of a blocking agent, "rff,is given by •o =
+ k+
[Bl]
(3)
96
Because the EPSC decay time constant, ~'d, becomes markedly shortened in presence of the AChR open-channel blocker (Fig. 2A1), one should expect that mean burst time, %, would likewise be shortened. This conclusion is what follows from theoretical predictions of the effects produced by open-channel blockers [15] if instead of fast-dissociating blockers, e.g. local anaesthetics, a slow-dissociating blocker, e.g. hexa- or heptamethonium, is used (for more detailed discussion on this point see [56]). The results obtained with heptamethonium on single AChR channel activity in rat superior cervical ganglion neurones [20] have confirmed this conclusion. Both r b and %2 are shortened by heptamethonium, the former much more than the latter. The 'apparent' k+ B value, k '+B, as estimated from an equation similar to (3) but with % and r£ (burst duration in the presence of a blocker) instead of % and %', yields a mean k '+ B value of 18 x 10 6 M - ] . s -1, while the mean k+~ value is 65 × 106 M -1 . s -I
[201. An easier way to obtain k~B is to replace in equation (3) % and %' with the EPSC decay time constants measured in normal ( r d) and in blocker-containing (~-d) solutions. The k'_ B value can be estimated as the inverse of the time constant that characterises the restoration of AChinduced current evoked at various intervals after the conditional ACh application (Fig, 2A 2)- From k+B and k ' B, the affinity constant K B' = k+' 8 / k '- B can be found. Fig. 2A 3 shows that there is no voltage-independent component in the blocking effect produced by hexamethonium on the ACh-induced current, as the blocking effect drops to zero at positive membrane potential levels. On the other hand, it was found both in parasympathetic [4] and in sympathetic [48] ganglia that the k '+ B value for hexamethonium is higher than that of tubocurarine. Because hexamethonium is a specific ganglionic blocker, in contrast to tubocurarine, it was suggested that this difference is due to the open-channel-blocking effect which in this case determines the selectivity of ganglionic blockade [48]. To check this suggestion, the k'+e and Ke values were determined for several specific ganglion-blocking agents and were compared with
]
;Ai[(;LION-~ILOCK!N; A~:'~[V2'
[ ] k'+B [] K'B
% 1000 1/30
10
O.I
O. Ol
IEH
C4
C5
C6
C,,.;.
P
11t9
Fig. 3. Comparison of ganglion-blocking activity with channel-blocking activity for ganglion-blocking drugs IEM1119, tetra-(C4), penta-(Cs), hexa-(Cr), hepta.methonium (C7) and pirilenum (P) as indicated at the abscissa [53,55]i
their potencies to block ganglionic transmission [55] (Fig. 3). The ganglion-blocking potency of all the agents tested correlates ( P > 0.95) with their t t k+B values (but not with their K B values). Therefore, the rate of binding of a blocker to an open channel is most important in the blockade of synaptic transmission [53 56]. The lack of correlation between the ganglion-blocking potencies and K~ values is a result of two factors; (1) the k values differ among the drugs used much less than the k~B values, and (2) the frequencms of the preganglionic stimuli applied to test transmission blockade are too low to detect significant differences in k'_ s values between the drugs used. A similar correlation between the open-channel-blocking and ganglion-blocking activities was later observed by Gurney and Rang [25] with the short-chain bis-quaternary ammonium compounds mentioned above, but not with long-chain bisquaternary ammonium compounds (longer than C7), which are not specific gan$1ion blockers. Most recent experiments revealed a new class of very specific blockers for synaptic transmission through enteric nervous plexuses which are char-
97 acterised by extremely long action [56]. The investigation of mechanisms of their action suggests that they combine the open-channel blocking effect with irreversible binding to the A C h R molecule (Gmiro, V.E., Derkach, V.A., Kurrenny, D.E. et al., in press). The second mechanism, the blockade of a recognition centre, is illustrated by the effect produced with trimethaphan, a specific ganglionblocking agent. Trimethaphan, unlike hexamethonium, decreases the amplitude of EPSC in sympathetic neurones without decrease in % (Fig. 2 B1) [7]. Another marked difference from hexamethonium is that the effect of trimethaphan on the ACh-induced current is frequency-independent (Fig. 2B2) and voltage-independent (Fig. 2B3); the membrane hyperpolarization is not followed by an increase in the trimethaphan-induced blockade. Thus, trimethaphan does not produce open-channel blockade. These results are consistent with earlier observations on the rat submandibular ganglion where trimethaphan is a pure competitive blocking agent [4]. Pure competitive effects on ganglionic AChRs have also been observed with a-neurotoxins, in particular, with surugatoxin [see 9], a-bungarotoxin and acobratoxin [47]. It can be concluded that specific ganglionic blockade produced by different drugs is due to a blockade of either the A C h R open channel or the AChR recognition center. The blockade of synaptic transmission through the blockade of the open postsynaptic channels, which may at first look surprising, is due to a decrease in the electric charge carried by the EPSC, which in turn causes a decrease in the amplitude of postsynaptic potential, thus making it subthreshold for the postsynaptic spike initiation [53,54]. The drugs which produce the specific openchannel blockade in nicotinic AChRs of symphathetic ganglion neurones, do this also in nicotinic AChRs of the neurones of parasympathetic ganglia and of molluscan neurones [3], but not in those of motor end-plates [32,44]. This difference correlates with the sensitivity of the A C h R channels to calcium ions that can prolong mean channel-open time (see above). One may suggest that the specific open channel-blocking drugs bind to
the sites in the channel that normally interact with calcium ions. This suggestion is consistent with the observation that increased external concentration of calcium ions strongly inhibits binding of hexamethonium to the open channel, as indicated by the marked drop in k '+8 in sympathetic ganglion neurones [50]. Moreover, calcium-binding sites have been found in the ionic channels of nicotinic AChRs (in molluscan neurones [11,57]). To summarise the characteristics of nicotinic acetylcholine receptors in autonomic ganglion neurones, one should emphasise that they are unique in at least 3 respects, when compared with nicotinic acetylcholine receptors in other tissues: (1) in the long mean channel-open time, both 'true' and 'apparent', which determines long-lasting postsynaptic current decay; (2) in the high sensitivity of the channel kinetics to increased concentration of calcium ions; (3) in the susceptibility of the open channel to drug-induced blockade, which underlies the specificity of many ganglionic blockers. It seems likely that all 3 characteristics relate to the same peculiarity of the channel-operating mechanism which possibly involves calcium ions.
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