TIPS - December 1987 [Vol. 81 321,406-411 9 Goldman, D., Deneris, E., Luyten, W., Kochhar, A., Patick, J. and Heinemann, S. (1987) Cell 48, 965-973 10 Hermans-Borameyer, I., Zopf, D., Ryseck, R. I’., Betz, H. and Gundelfinger, E. D. (1986) EMBO ].5,1503-1508 11 Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., Asai, M., Inayama, S., Miyata, T. and Numa, S. (1982) Nature 299, 793-797 12 Claudio, T., BaBivet, M., Patrick, J. and Heinemann, S. (1983) Proc. Nut1 Aced. Sci. USA 80,1111-1115 13 Devillers-Thiery, A., Giraudat, J., Bentaboulet, M. and Changeux, J. P. (1983) Proc. Nut1 Acud. Sci. USA 80,2O67-2071 14 Guy, H. R. (1984) Biophys. J. 45,249-261 15 Finer-Moore, J. and Stroud, R. M. (1984) Proc. Natl Acud. Sci. USA 81,155-159 16 Lindstrom, J. (1986) Trends Neurosci. 9, 401-407 17 Giraudat. I.. Montecucco. C.. Bisson. R. and Char&x, J. P. (1985) Biochemistry 24,3121-3127 18 MacCrea, P. D., Popot, J. L. and Engebnan, D. M. (1986) Biophys. J. 49, 355a 19 Dunn, ‘3. M. J., Conti-Tronconi, B. and Raftery, M. A. (1987) Biochem. Biophys. Res. Commun. 139,83O-837 20 Karlin, A. (1980) in The Cell Surface and Neuronul Function (Poste, G., Nicolson, G. L. and Cotman, C. W., eds), pp. 191-260, Elsevier 21 Tzartos, S. J. and Changeux, J. P. (1984) J. Biol. Chem. 259,11512-11519 22 Mishina, M., Kurosaki, T., Tobimatsu, T., Morimoto, Y., Noda, M., Yamamoto, T., Terao, M., Lindstrom, J., Takahashi, T., Kuno, M. and Numa, S. (1984) Nature 307,604-608 23 Culver, P., FenicaI, W. and Taylor, P. (1984) J. Biol. Chem. 259,3763-3770 24 Kurosaki, T., Fukuda, K., Konno, T., Mori, Y., Tanaka, K., Mishina, M. and Numa, S. (1987) FEBS Lett. 214,253-258 25 Kao, P., Dwork, A., Kaldany, R. Silver, M., Wideman, J., Stein, S. and Karlin, A. (1984) J. Biol. Chem. 259,11662-11665 26 Neumann, D., Barchan, D., Safran, A., Gershoni,J. M. and Fuchs,S. (1986) Proc. Nutl Acad. Sci. USA 83, 3008-3011 27 Mishina, M., Tobimatsu, T., Imoto, K., Tanaka, K., Fujita, Y., Fukuda, K., Kurasaki, M., Takahashi, H., Morimoto, Y., Hirose, T., Inayama, S., Takahashi, T., Kuno, M. and Numa, S. (1985) Nature 313,364-368 28 Dennis, M., Giraudat, J., KotzybaHibert, F., Goeldner, M., Hirth, C., Chang, J. Y. and Change& J. P. (1986) FEBS Lett. 207,243-249 29 Neher, E. and Steinbach, I. I-I. (1978) 1. .. Physiol. (London) 277,15<176 30 Cohen, J. B., Weber, M. and Changeux, J. P. (1974) Mol. Pharmacol. 10,904-932 31 Heidmann, T., Oswald, R. E. and Changeux, J. P. (1983) Biochemistry 22, 3112-3127 32 Changeux, J. P., Pinset, C. and Ribera, A. B. (1986) J. Physiol. (London) 378,497513 33 Heidmann, T. and Changeux, J. P. (1986) Biochemistry 25, 6109-6113 34 Cox, R. N., Kaldany, R. R., DiPaola, M. and Karlin, A. (1985) J. Biol. Chest. 260, 7186-7193 35 Giraudat, J., Dennis, M., Heidmann, T., Chang, J. Y. and Changeux, J. P. (1986) Proc. Nut1 Acad. Sci. USA 83,2719-2723 36 Giraudat, J., Dennis, M., Heidmann, T., Haumont, P. T., Lederer, F. and
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Changeux, J. P. (1987) Biochemistry 26, 2410-2418 Hucho, F. L., Oberthtir, W. and Lottspeich, F. (1986) FEBS Lett. 205,137-142 Herz,: M., Johnson,D. A. andTaylor, i. (1987) J. ~tot. Chem. 262, 7238-7247 Imoto, K., Methfessel, C., Sakmann, B., Mishina, M., Mori, Y., Konno, T., Fukuda, K., Kurasaki, M., Bujo, H., Fujita, Y. and Numa, S. (1986) Nature 324,67O-674 Furois-Corbin, S. and PuBman, A. (1986) Bfochim. Biophys. Actu 860, X5-177
41 Monod, J., Wyman, J. and Changeux, J. P. (1965) J. Mol. Biol. 12,88-118 42 Jackson, M. (1984) Proc. Nutl Acad. Sci. USA 81,3901-3904 43 Changeux, J. P. and Heidmann, T. (1987) in New insights into synaptic function (Edeiman, G., GaB, W. E. and Cowan, W. M., eds), pp. 549-604, John Wiley and Sons 44 H&felt, T., Holets, V. R., Staines, W., Meister, B., Melander, T., SchaBing, M., Schulzberg, M., Freedman, J.,Bjiirklund, H., Olson, L., Lindh, B., EIfvin, L. G., Lundberg, J. M., Lindgreen, J. A., Samuelsson, B., Pemow, B., Terenius, L., Post, C., Eve&t, 8. and Goldstein, M. (1986) Prog. Brain Res. 68,33-70
45 Boyd, N. and Leeman, S. (1987) J. Physiol. (London) 389, 69-97 46 Miledi, R. (1980) Proc. R. Sot. London Ser. B.209,447452 47 Takeyasu, K., Shion, S., Udgaonkar, J- B.. Fujita, N. and Hess, G. P. (1986) Biochemistry 25,1770-1776 48 Huganir, R. L., Delcour, A. H., Greengd, P. and Hess,G. 6. (1986) Nature 321.744-776 49 Revah, F., MuBe, C., Audhya. T., Goldstein, G. and Changeux, J. P. (1987) Proc. Nat1 Acud. Sci. USA S4,34n-3481 50 Fischer, J. B. and Olsen, R W. (1986) in Biochemical aspects of GABA/benwdiuzepine receptor function, PP. 241-259, Alan R. Liss 51 Ascher, P. and Nowak, L. (1987) Trends Neurosci. 10,2B4=288 52 Grenningloh, G., Rein&, A., Schmitt, B., MethfesseI, C., Zensen, M., Beyreuther, K., Gundelfinger, E. D. and Betz, H. (1987) Natxre 328,21!%?20 53 Schofield, P. R., Da&on, M. G., Fujita, N., Bunt, D.R., Stephenson, F.A., Rodriguez, H., Rhee, L. M., Ramachandran, J., ReaIe, V., Glencorse, T. A., Seeburg, P. H. and Barnard, E. A. (1987) Nature 328,22X227 54 MacCrea, P. D., Popat, J. L. and Engehnan, D. H. EMBO J. (in press)
Nicotinic acetylcholine receptors of nerve and rmscle: functional aspects David Colquhoun, David C. Ogden and klistair Mathe Cloning studies suggest that there are at least five types of nicofinic receptor with different amino acid sequences. David Colquhoun and colleagues discuss pharmacological consequences of this heterogeneity. The function of the muscle type receptor has been examined in some detail, though if is only recently that the equilibrium concentration-response relationship has been determined reliably. Much less is known about neuronal nicotinic receptors, which have sequences (and probably subunit sfoichiomefry) that differ considerably from those of the muscle and electric organ types. They also differ considerably from the latter in their abilities to bind various toxins. The usual nicotinic antagonists, such as tubocurarine and hexamethoni_um, show great variability between receptor type, and between species, in the extent to which they work by competitive block or by block of the ion channel. It is often said that more is known about the nicotinic acetylcholine receptor (nAChR) than about any other. It may therefore be salutary to start by listing some of the things that are not known about it (see Box 1). The nAChR should not, of course, be referred to in the singular. It has been known at David Colquhoun is Professor of Pharmscology and Alistuir Mathie is u Postdoctoral Fellow in the Department of Pharmacology, University College London, Gower Street, London WCZE 6BT, UK. Both ure members of the MRC Receptor Merhaaisms Research Group.David Ogden is u Lecturer in the Department of Pharmacology, King’s College, Strand, London, WC2R 2LS. UK.
least since the work of Paton and Zaimis’ that there are great differences between the nACllR of the skeletal muscle endplate and those on the postsynaptic neurones of autonomic ganglia. Furthermore most muscles can make embryonic and adult forms of the receptor that differ in structure. The-receptors of electric organs of the skate and eel are different from muscle and ganglionic receptors, and, much more recently, recombinant DNA techniques have suggested the presence of at least two more sorts of the nAChR in the brain.
Q 1987. Elsevier
Publications,
Camhridw
0165 - 6147/77/602.~
TIPS - December
466 To add to the complications it
seems that there are considerable differences between the nAChR found in sympathetic neurones of frog and rat (see below). Even within one species we must now think in terms of at least five nAChR (two muscle and three neuronal) all with different primary structures. lit seems that there is an extended family of genes that code for sevxzl types of nicotinic receptor channel. There is 80% Sequence homology between the at-subunit of the receptor from Toledo ektric organ and that from human muscle, but neurones of autonomic ganglia and the CNS contain receptors that appear to be more divergent members of the same family. The muscle nicotinic receptors It is the long-term aim of pharmacologists to relate the function of receptors to their structure. Although there have recently been big advances in both areas, the final links remain to be forged. s)ructtlre l2ndfWlcsion Muscle and electric organ receptors all have four sorts of (structurally-related) subunits in an a$yS pentamer (e.g. Ref. 10). In the adult form of the calf receptor the embryonic y subunit is replaced by an E subuni?.
There has been much discussion about how the cfiains of each subunit cross the membrane3. It has been suggested that an amphipathic region (the MA segment) of the chain might form the lining of the ion channel. However this appears to be contradicted by the first real investigations of the relationship of structure and function by alteration of the structure of the receptor (rather than that of the agonist or the permeant ion); Imoto ef aL4 have found evidence that a region near the hydrophobic M2 segment appears to be strongly involved in ion permeation, a view that is supported by work with certain channel blockers5”. There is, so far, no real progress in solving the most basic of pharmacological problems, viz. defining the difference between shut, open and desensitized forms of the receptor, and hence underst~d~g how agonists alter the equilibrium between these states. The eguzX&tiulncuncen~u#iorrresponse cwve for acetytchotine The most fundamental response that can be measured is the fraction of channels that are open (at a given moment) as a function of agonist concentration. The existence of rapid and profound desensitization has made the measure-
Some things that are not known about dcotinic receptors 1 Secondary and tertiary structures, location of membrane crossing segments
2 3 4 5
6
Difference between structures of shut, open and desensitized states Subunit stoichiometry for all neuronal receptors Nature of the ion charmei lining
Carbohydratestructure and function Difference between adult and embryonic form sequences (except for
calf); their relation to the ‘denervated’ form and their control ? ?recise sites of agonist aad antagonist binding
Function 1 Equilibrium concentration-response (Popen)curves (corrected for desensitization), except for frog junctional and BCWI-cell receptors; not known for any neuronal receptor8 and not Edenknown in frog for many agonists (e.g. is decamethoniitm really a partial agonist?) 2 Nature and meaning of the ‘fine structure’ (short gaps etc.) of channel openings for ali neuronal and for many other receptors 3 Channel opening rates and microscopic agonist affinities for most receptors except (perhaps) frog and snake mtiscle and BC3Hl cell-line 4 The extent of non-equivalence of the two rw-subunits 5 The rate constants for association and dissociation of competitive
a@lgoiliStS 6 The nature of ‘non-simple’channel block (except for ‘trapping’)
1987 Wut.
81
ment of this relationship near impossible until recently, though some good attempts were made (see Ref. 11). Fig. la shows the response to a high concentration (200 pi) of ace~lcho~ine at a frog muscle end-plate. The current that flows through all of the lo7 or so channels is shown, and this current will be directly proportional to the fraction of channels that is open at any moment. The peak current is over 16 PA, which corresponds to opening of almost all of the ion channels present, but the response desensitizes, rapidly at first and then more slowly, until it is only 1. or 2% of the peak value. In a singIe channel record the response must be interpreted as the fraction of time for which the channel is open, i.e. the probability of being open, denoted Popen. Fig. lb shows the single channel response to 200 PM acetylcholine (the same concentration as for the response in Fig. la). Openings occur in rapid succession because of the high ccmcentration (about 230 activations of the receptor per second, each lasting, on average, about 4 ms with only about 0.2 ms shut time between activations). But the clusters of high activity (which last for several hundred milliseconds on average) are separated by long silent periods during which all the channeIs in the patch are desensitized’. By cutting out these silent periods, and estimating Popen only during the periods of high activity, a concentrationresponse curve can be constructed from which the effects of desensitization have been eliminated. The result’ is shown in Fig. 2. At negative membrane potentials the curve rises to a maximum Popen of 0.9 at 100 PM acetylcholine but then falls again, despite the fact thai desensitization effects have been eliminated; this decline has been shown to be the result of block of the open ion channel by acetylcholine itsel8. The affinity for the channel blocking reaction is quite low (KB= 1.2 mM at -110 mV; the mean duration of a blockage is only about 20 ps). After allowing for the effect of channel block the results suggest that acetylcholine is capable of opening about 98% channels at high concentration, i.e. it is a very efficacious agonist, the equilib-
TIPS - December
1987 [Vol.
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467
a
2OOpn4 acelylcholine-50mV
s
200~ acelylchdine -116mV
7
-
5vA
218s
111-r-
FQ. 1. (a) Currentfhmugh a frog nwecle endplate (in a vaselinegap voltage clamp) evoked by application of acelykhohe (200~). Membrane potential -5OmV. T =4”c. Reproduced
1926
muscleendp&te. The &aces shown are perts of a continwus record, the kfgih b$gtpAlhe
ofthe Mentpedod end
fromCachelin.A. 8. (1997) PhD lhesis,University of London.(b) Sing~annel cutrentsevokedby acetykholine(200 m) in fmg
rium constant for the opening reaction once the agonist sites are occupied (b/a, see Box 2) being about 30. The lower curve in Fig. 2 shows the concentration-response curve at positive membrane potentials. The block is strongly dependent on membrane potential; it is even weaker at positive membrane potentials (Ka = 30 mM at +lOO mV). Virtually no decline of the response is seen at high concentrations now. However, the maximum response is only about 41% open, so acetylcholine is a partial agonist at positive membrane potentials (p/a! has fallen to about 0.7, largely because the mean open channel lifetime, l/a, is much shorter when the cell is depolarized). In pharmacological terms (e.g. Ref. 11) the ‘efficacy’ of acetylcholine is dependent on membrane potential (whereas the affinity for the binding reaction is much less so).
The rate ofaction of acetylcholine The discussion so far has centred on the equilibrium response to acetylcholine, but synaptic transmission is a rapid process too fast for equilibrium to be reached; to understand it we need to know the rates of action of agonists and antagonists. The first attempt to measure the rate of association and dissociation of tubocurarine from the receptor was made by A. V. Hill”. He concluded, incorrectly as it turns out, that the rates he measured were not diffusion controlled. Many other attempts to mea-
b
t
sure rates for competitive antagonists since then have also met with little success (see Ref. 11) although equilibrium constants have been measured quite accurately. The use of single channel methods may now allow this longstanding problem to be solved. The methods of noise and single channel analysis allowed a start to be made on the rate of agonist action. They provided estimates of the mean lifetime (l/or) of the open ion channel, and hence of the rate constant, 01, for channel shutting (see Box 2). The rate constants for channel opening, and for agonist association and dissociation, have proved far less accessible. In principle these quantities can
_ Acetylcholine
between swcessive traoes Reproduced frcnn Ref. 8,
ofeach record.
be estimated from observations of the fine structure of channel activations; a single activation of the channel by agonist usually pmduces several openings in quick succession (the nachschlag phenomenon) rather than a single opening. The extent to which such multiple openings occur should depend on the values of the opening rate constant (6, see Box 2) and of the agonist dissociation rate constant (k-2); the relative value of these rates controls how likely it is that &e doublyoccupied shut channel will open (or re-open), rather than return to the resting state via agonist dissociation’3*‘4. Observations on junctional receptors from frog
.
-12C)mV (mean)
0 4 :XWiV (maan) i
SEU
1.0 -
0.8 0.6 Plopenl
Concentration(p) fig. 2. ResponsefP_)
of frog endplatechannelspronedagainsl acetylchdine concentration (kg scale)at two differentmembranepolentiats;upper CUNB, -95 to - 130mV; lowercurve,+85 lo + 120 mW.Reproduced fromRef. 9 withpermission.
TIPS - December 1987 Wof. 81
468
muscle15, when interpreted in this suggest that for acetylcholine (at -130 mV and 12”(Z), 1/4X= 1.4ms, $=30600 s-’ and k-r== 8150 s-l. Thus @/or= 43, the ‘efficacy’ of acetychohne is high; it is pred.cted that 98% (i.e. 43144) of channels will be openable by high acetylcholine concentrations, which is consistent with observations (Fig. 2). The affinity of acetylcholine for its binding site, on the other hand, is quite low, the equilibrium constant being about 80 pru. These values are near-ideal for rapid synaptic transmission’5. They predict that acetylcholine will produce 50% receptor occupancy at a concentration of about the combined 12 !A& though effects of desensitization and channel block have made this number impossible to determine in direc! binding experiments. The large value of the opening rste constant 6 implies that there will be about 30000 opening transitions per second of time spent in the doubly-occupied shut state, and this,. combined with the rapid dissociation rate (net rate 2k_, = 16300 s-i) means that this state will have a very short lifetime (about 20 us). The results also imply that neither the agonist binding step nor the shut-open conformation change step can be treated as rate limiting; they have comparable rates. The rapid opening rate is about what would be expected from the fast rate of rise of miniature endpiate currents that is observed at the neuromuscular junction16. On the other hand experiments with the ‘embryonic’ type of nicotinic receptor of the BC3H-1 cell line have, when interpreted as above, suggested an opening rate fi almost 100 times slower than that for frog junctional receptors (and a dissociation rate about 10 times slower), though, because the open time is much longer for these receptors, the inferred values of g/cx, i3.7, was still quite large”*‘*. way,
Agonistsother than ace~lc~o~ine Work similar to that described above has suggested that the fact that carbachol is about 21-fold less potent than acetyicholine results mainly from its lower affinity for the receptor, rather than from its (- 2-fold) Iower efficacy once bound”. Suberyldicholine, on the
other hand, is about 3.5-fold more potent than acetylcholine, despite having a lower efficacy, because of its higher affinity for the receptoP. AR agonists tested have proved to block the ion channel; in fact acetylcholine itself produces less block, for a given degree of activation, than any other agonist that has been tested. Suberyldicholine produces much stronger block’, and most putative partial agonists such as decamethonium2’ produce such strong ion channel block that it has not yet proved possible to tell whether they appear to be partial agonists merely because of their self-blocking actions, or also have a whether they genuinely low ability (low p/o) to open channels (Ref. 21, and Marshall & Ogden, unpublished observations). !t has been suggested that acetykholine produces an additional, powerful inhibition of its own action (‘isosteric inhibition’) by binding to some separate site even when the channels are shup. This work was on receptors of electric organs; the results differ from those in frog muscle in which most of the ion channels can be opened by acetylcholine (Fig. la), and appear to be inconsistent with current ideas concerning the margin of safety of neuromuscular transmission. Antagonists at the receptor and ion channel The classical non-depola~zing antagonists such as tubocurarine and gallamine act predominantly by competition with acetylcholinc under physiological conditions. This action is not dependent on membrane potential. The a-toxins such as rr-bungarotoxin also combine near-irreversibly with the agonist binding site in both mammalian and frog muscle receptors. In addition all the antagonists that have been investigated (which is relatively few of them) also show a voltage-dependent channel blocking action (see Ref. 11). For tubocurarine and gallamine this effect can be quite important under certain experimental conditions. There are many other compounds that work predominantly by ion channel blocking e.g. barbiturates and ‘local anaesthetics’ (see Refs 11,23-25). These channel blocking effects frequently do not obey the simple mechanism shown in Box 2
(see Refs 11,23-26). This mechanism predicts, for example, that the total open time per channel activation should be unaltered by the blocker which often appears not to be the case. This appearance could occur: (1) if there existed additional long-lived blocked states distal to that shown in Box 2; or (2) if there existed a route from the blocked channel back to the resting state that did not involve reopening of the channe19,‘q*26. Some blockers may also be able to work whether the channel is open or not. Little is known of the mechanisms of these deviations from simple open channel block (except for the case of trapped antagonists which are discussed below). The neuronal nicotinic receptors The difference between muscle and neuronal receptors shown by Paton and Zaimis’ was emphasized by the discovery that most neuronal receptors are not blocked by cu-bungarotoxin, which blocks the muscle type nearly irreversibly, whereas other toxins (e.g. surugatoxin, K-toxins) block at least some neuronal receptors far more effectively than muscle receptors (see Refs 27 and 28). These differences now have a structural basis. The muscle-type (Ysubunit has been called the u-l subunit. An ru-2 subunit has been identified as a gene in chick and rat. An ar-3 subunit has been cloned from the PC12 cell line (derived from rat phae~hrom~ytoma cells); it is expressed in various . rat of the br&?s30. zyzst recently an a4 subunit has been cioned3* which is also expressed in the brain, and which can give rise, by alternative splicing of the same primary transcript, to two different mRNAs, or-P1 and o-4-2. The 1x-3 subunit has only 47% homology in amino-acid sequence with the mouse muscle w-1 subunit (and 58% nucleotide sequence homology), though this figure conceals a much greater homology in the putative membrane-spanning regions (66887% in Ml to M4). The four cysteine residues (at positions 128,142,192 and 193 in muscle), which characterize all ar subunits, are present in the ar-3 neuronal subunit. However, much of the extracellular sequence near them, which is thought to contain the binding
TIPS - December 1987 lVol.81
-Box 2-
A simple reaction scheme for the nicotinic receptor A simple scheme that can account for
most, thoug’imiiot all, observations on muscle nicotinic receptors is shown diagrammatically in the figure (a). It is supposed that two agonist molecules (shown as @) bind sequentially to two sites on the (Ysubunits, after which a conformation change from the shut to the open state may occur. The open state may then be blocked by the agonist itself, or by many antagonists (shown as +). This scheme excludes desensitization, trapping (and other complex behaviour) of some channel blockers, possible non-equivalence of the (Y subunits, and the possibility that the singly-occupied channel may also open.
a
SHilT
E+!Jfj& vacant
1bOlJd
b
nachschfug).
The effect of channel blockages will be to produce
Neuronal receptors in peripheral ganglia Both normal synaptic transmission and the effects of drugs differ in many ways from the neuromuscular junction. Effects of antagonists and toxins. Nicotinic receptors in peripheral ganglia of mammals and chick
1
W
Transitions between states occur, of course, randomly. In the figure, b shows an example of transitions that might occur between the four states :!E$JJ!L?jI C shown in a (excluding, for simplicity, the blocked state), whereas c shows what would actually be seen (the three shut states not being distinguishable on the experimental record). There will be single, and some double, occupancies that produce no opening at all. After an opening the receptor will return to the doubly-occupied shut state from which it may re-open (twice and once in the two activations shown) to produce a burst of openings (an ‘activation’), the openings being separated by brief shut periods. More rarely it could return to the singlyoccupied shut state before re-opening, so producing a longer gap within a burst. The mean number of short gaps per burst will be fVZk+, and their mean duration will be 1/@+2k_s). If such brief gaps can be correctly identified in experimental records, then values of j3 and k-2 can be inferred from them. The bursts, or activations, shown above might appropriately be called N-bursts (for
sites for agonists and bungaro,oxin, differs greatly between muscle and neuronal receptors; for example, of the 14 amino acids between 153 and 172 only one is the same in o-1 (muscle) as in o-3 (X12), and 8 of the differences involve replacement of uncharged by charged residues. This may explain the different toxin binding characteristics of the rat neuronal receptor.
SHUT
further interruptions of the channel activation, the mean length of a blockage being l/k+ Bursts so produced could be called B-bursts (for blockuge).The mean length of individual openings (whether or not they happen to be terminated by a blockage) will be shortened from l/or to ll(o+k+srs) where xa is the free concentration of biocker. Furthermore if we denote the mean length of the N-burst (more exactly, the open time per N-burst) in the absence of blocker as 11~ (the effective open tune for physiological purposes), this will be shortened by the blocker, just as the individual openings are, to l/&a + k+Gs) (see Refs 9, 14 for details). For a simple open channel blocker, hOWe-JET, the number of blockages (and hence the number of openings, or N-bursts) per B-burst will be such that the total open time per B-burst is unchangedby the blocker.
(also chromaffin cells and PC12 cells) are not blocked by o-toxins The such as o-bungarotoxin. related toxins rc-bungarotoxin and x-flavitoxin are highly effective at blocking nicotinic receptors in chick ganglia; trimetaphan (but a-bungarotoxin) protects not against this block. Toxin F, and probably Bgt 3.1, are identical with k-bungarotoxin2s,35. Kappatoxins also block mammalian ganglionic receptors, though with IO-fold lower potency28. The classical blocker hexamethonium was, for a long time, supposed to compete for the ganglionic receptor on the grounds that it did not produce depolarization. Blackman 2 suggested that hexamethoni ;Im might block the ion channe! lather than the receptor and this suggestion has proved to
be true in, for example, the rat submandibular ganglion33. Tubocurarine and decamethonium also appear to work largeiy by channel block in mammalian ganglia (which are not depolarized by decamethonium), whereas trimetaphan is more likely to produce genuine competitive block. Hexamethonium owes its high potency, and pronounced usedependence, to the fact that not only can it block the open channel, but the channel can also shut around the blocking molecule and trap it lmtil such time as the channel is opened again by acetylcholine. Larger molecules (e.g. decamethonium) cannot be trapped in this way and so are less potent34. Trapped channel blockers have not yet been characterized by single-channel analy-
TIPS - December 1987 [Vol. 81
r~.s(a)~Enannef~~~~~bys~~~~~
ina cWattachadpalch on a rat sympathat&neurone.Membrane potentiat was If,,, = (v, -50) mV where V,, is the rssting potentfaf of the callsos!My about -50 mK EandwidthDC-2 Wz.
T=~.@)~oftiFe~~~of~~~activations (rwnts) ofcha!Uve& evokedby acetylcholine(3 ~JzeWtached patch; Vnl= (v,, -50) mV. Dashedbox shows the number of sis, but the existence of two distinct blocked states, the distal ‘trapped’ state having a long lifetime, could explain’ the apparent (in this case not genuine) reduction in the total open time per channel activation that is a;common form of deviation from the behaviour expected of the simple o-n channel block shown in Box 2. AU the antagonists discussed above have actions in mammalian ganglia that differ considerably from those on muscle receptors. Frog ganglionic receptors, however, differ in many ways from those of mammals. They are blocked by obungamtoxin (though less effectively than muscle receptors), tubocurarine is predominantly competitive, and hexamethonium is very ineffective [Lipscombe, D. (1986) PhD thesis, University of London], as in muscle. Decamethonium shows signs of channel block in frog as well as rat ganglia, but it is also a weak agonist in frog ganglia [Lipscombe, D. (1986) PhD thesis, University of London]. Both of these effects resemble those in muscle. Thus frog ganglionic receptors resemble those of muscle more closely than do mammalian ganglionic receptors, although trimetaphan seems to have a greater competitive effect on both frog and rat ganglionic receptors than on muscle. ~ocu~izutjo~ of fork bilging. Ganglionic neurones bind ar-
exporie&G components(sotid i&e). TIme constantswere 0.8 ms and 11.0 ms wiltsareas of 47% and 53% respectively,Thereare marry short (O.Sms) openings but 97% dr the charge flows thy tha long (tl ms) activations which are therefore of primary importance for synapfic transmission.Modified from Ref.
42 with permission.
toxins despite their ineffectiveness as blockers. For example the chick ciliary ganglion binds about 20 fmollganglion of ar-bungarotoxin (with K=l nM). However, the bound molecules are mainly extrasynaptic, and, although the binding sites probably have structures very like nicotinic receptors, their physiological function, if any, is unknown. On the other hand Toxin F (K-bungarotoxin) which does block, is bound to the extent of only about 1 fmollganglion (K = 5nM), but its binding is much more localized to synaptic regions, there being about 600 sites l&rnW2 at postsynaptic thickenings on ciliary neurones’s*36~37.This density is far lower (roughly 20-fold) than at the muscle endplate. Channel densities have still not been determined for mammalian ganglionic neurones. Synaptic transmission. A lower channel density in ganglia than in muscle would contribute to the smaller conductance increase produced by a transmitter packet (quantum) in the former. In both chick ciliary ganglion and rat submandibular the ganglion quanta1 conductance change is roughly 20-fold smaller than in rat muscle38,39. The quantum opens roughly 100 channels in the ganglia but 100~2000 channels in muscle, although the depolarization produced is comparable in both, because of the much smaller size of the ganglionic cells. Stimulation of a preganglionic neurone
may release over 100 quanta (similar to the value of motoneurones) onto singly-innervated cell5 of rat submandibular ganglion, but only 1-3 quanta onto a guinea-pig superior cervical ganglion cell which is innervated by u to 15 preganglionic neurones3B*B . Channel properties in ganglia. The single channel currents shown in Fig. 3a look similar to those in muscle. However, the single channel conductance in ganglia is generally smaller than in muscle of the same species. In rat sympathetic neurones it is 35 pS at 20°C with 1 mM Ca’+ (Ref. 42; cf. Derkach ef al.‘3 who found only 20 pS, possibly because of their use of a higher Ca*+ concentration, and Tris buffer} and it is similar in rat submandibular ganglion. Cultured chick ciliary ganglion gave 40 pS at 3O”C?*.On the other hand mammalian endplate receptors have a conductance of 70 pS (e.g. Ref. 45). In frog ganglion Lipscombe fZoc. cif.) found 18.5 pS at 20°C whereas frog end late channels are 30 pS (at 10%J4 H. Rat sympathetic neurone channels also show a pronounced increase in noise level when they are open (Fig. 3a)4*c42.Single channel activations are interrupted by brief shut periods as in muscle (Fig. 3a), but their analysis, along the lines shown in Box 2, has barely begun. The mean duration of a single activation (‘burst’), about 12 ms at 2oOC as illustrated in Fig. 3b, is simildr to the time constant for
TlPS - December 1987 iVol. 81 decay of nerve-evoked postsynaptic currents in a number of mammalian ganglia (see Ref. 41 and references therein). This suggests that ace~lcholine is eliminated rapidly from the synaptic cleft as in muscle. The mean burst length for muscle channels is much shorter, 1-2 ms at 2ffC. Areas of uncertainty. Much remains to be known about many aspects of ganglionic transmission. For example little is known about the postsynaptic receptor density, or the disposition of released transmitter, in mammalian ganglia. Not only the kinetics but also the equilibrium concentration-response curve have yet to be adequately defined for any neuronal receptor (cf. Fig. 2); preliminary results Ahow a puzzling heterogeneity in Popenbut no gross difference from muscle in the potency of acetylcholine in activating single channels (A. Mathie, unpublished data). The very prorectification inward nounced shown by ganglionic channels has also yet to be explained satisfactorily42. In the mammalian CNS, as in ganglia, there are many binding sites for ff-neurotoxins but in most cases that have been tested (including the Renshaw cell in the spinal cord) a-toxins block neither synaptic transmission nor the response to acetylcholine (see Ref. 28). The rat brain also contains sites which bind both nicotine and acetylcholine with high (K= 220 nM) affinity (see Refs 47, 48), which have a different topographical distribution in the brain from that for a-toxin binding. The distribution of high affinitv nicotine and acetylcholine ginding sites is, however, similar to the distribution of RNA homologous to the a-Q-1 clone discussed above, as determined by in-situ hybridization to rat brain sections radiolabelled anti-sense usin RNA‘3*. The medial habenula, thalamus, hypothalamus and cortex all hybridize strongly. It is not yet known whether this means that all high-affinity nicotine-binding sites have (u-4subunits. The distribution of (u-3 gene expression, similarly determined, is substantially different from that oi a-4 (except for the medial habenula which hybridizes strongly with both)30. Conversely FYI2 cells,
47i from which the a-3 clone is derived, do not con&in highaffinity acetylcholine binding sites49. Clearly there is more than one sort of nicotinic receptor in the CNS. It is thought that some may be presynaptic rather than postsynaptic (the former could perhaps be a-3 and the latter a-4; Ref. 31). Little is known of the physiological role of either. Neither is the subunit stoichiometry of neuronal receptors known. Another rat brain receptos”, the relationship of which to the ~1-3and or-4types is unknown, has been suggested to consist of only two sorts of subunit, perhaps &MB)2 or f&@j~. And an insect nicotinic receptor may have two or even only one type of subunit (see Ref. 51 for discussionj. Classification of nicotinic receptors In the past there has been a strong tendency to base receptor classification on the characteristics of the binding sites for competitive antagonists, largely because the affinities of such antagonists could be determined reliably, by the Schild method (for example see Ref. 52). (Classifications based on agonist potencies, though theoretically much more dubious, have also given sensible results.) If this were still the case there would be a major problem in defining nicotinit receptors because antagonists which are primarily competitive on some receptors (e.g. tubocurarine on muscle receptors) are not competitive but work on the ion channels in other nicotinic receptors (e.g. in mammalian ganglia), or have mixed effects. Furthermore the very hiah affinity bindin sites for nicotine presumably cI?aracterize desensitized rather than native receptors and it is possible that the extent of desensitization may depend on, for example, the extent of phosphorylation of the receptor and hence on the experimental conditions (see Ref. 53). However, it is now clear that it is not sensible to define nicotinic receptors solely in terms of their agonist and antagonist binding sites. They must ultimately be defined in terms of the structure of the entire receptor-ion channel molecule, by means of recombinant DNA methods. Until this is completed, the use of toxins and antagonists will undoubtedly
remain useful, but the variability in their basic mode of action from one sort of nicotinic receptor to another is a potential source of confusion that must be borne in mind. References 1 Paton,W. D.M. and Zaimis, E. J- (1951} Br. 1. Phannacol. 6.155-168 2 Miihina, M., Takai, T., Imoto, K., Noda, M., Takahashi. T., Numa, S.. Methfessef,C. and sakmann,8. (1986) Nutare
321,40&411
3 Guy, H. R. and Hucho, F. (1987) Trends Neurosci. 10,318-321 4 lmoto, K, M&f&&, C, Sakmann, B., Mishina, M., Mori, Y.. Kom~o, T., Fukuda, K., Kurasaki, M., Bujo, H., Fujita, Y. and Puma, S. (:986) Nahre 324,670-674 5 Giraudat,. I., Dennis, M., Heidman. T., Chang, J. Y. and Changeux, J-P. (1986) Proc. NaN Acad. Sci. USA 83,2719-2723 6 Hucho, F., Oberthiir, W. and Lottspeich, F. (1986) FEBS Left. 205,137-X2 7 Sakmann, B., Patiak, J. and Neher, E. (1980) Nature 286,71-73 8 Colquhoun, D. and Ogden, D. C f. Physiof ELondoni (in press) 9 Ogden, D. C. and Colquhoun, D. (1985) Proc. R. Sot. London Ser. B 22!?,329-355 10 Maelicke, A. (ed.) (1986) Nicotirzic A~~lcholi~ Rccepfot Sfrucfure and Function (NATO AS1 series H, Vol. 3) Springer-Vedag. 11 Colauhoun. D. (1986) in Handbook of’ E~~rn~f~l P~o~~oio~ Vol. 76. (Kharkevich, D.A., ed.). pp. __ 5%113. Smineer-Verlae: 12 I&U, z;. V. (190$ I. PhysioJ. fLondonl39, .%xX73 ““” _.” 13 Colquhoun, D. and Hawkes, A. G. (19771 Proc. R. Sot. London Ser. B 119, i31-i62 14 Colquhoun. D. and Hawkes, A. G. 11983) in Sir& Channef Recording (Sak&&I, 8. &d Neher, E., ed& pp. 135-175, Plenum Press 15 Colquhoun, D. and Sakmann, B. (1985) I. Phys~ol. (Londonf 369,.501-557 16 Land, 8. R., Salpeter, E. E. and Salpeter, M. M. (1981) Proc. Natl Acad. Sci. USA 78,7200-7204 17 Sine, S. M. and Steinbach, J. H. (19%) I. Physiol. (London) 373,129-162 18 Sine: S. M. and Steinbach, J. H. (1987) !. Phusiol. (London) 385,325-360 19 ‘Ma&hall, C. G. and Ogden, D. C. (1986) Br. J. PharmncoI. 87,140P 20 Castillo, J. del, and Katz, B. (1957) Proc. R. Sot. London Ser. B 146,36+381 21 Adams, P. R. and Sakmann, B. (1978) Proc. Nuti Acad. Sci. USA 75,2994-2998 22 Udgaonkar, J. B. and Hess, G. I’. (1987) Trends Pharmacol. Sci. 8, 190-192 (see also ibid. 294-295 and 335) 23 Colquhoun, D. (1981) in Drag Receptors and their EfJectors (Birdsall, N. J. M., ed.), pp. 107-127, Macmillan 24 Adams, P. R. (1981) f. Membr. Biol. 58, 161-174 25 Rper, K., Bradley, R. G. and Dreyer, F. (1982) Physiol. Rev. 62,1271-1340 26 Neher. E. (1983) 1. Phvsiol. lLondonl339,
663-698 --- -._
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27 Brown, D. A. (1979) in Advances ia Cytopharmarofogy, Vol. 3, Neurotoxins (Ceccarelli, 8. and Clement, F., eds), pp. 225-230. Raven Press 28 Chiappinelli, V. A. (1985) Phrtf~aco~.
TIPS --December 7Iter. 31,132 29 Boulter. I.. Evans. K., Goldman, D., Martin; G.; Tmco, D., Hebwmann, S. and Patrick, J. (1986) Nafure 319, 36% 383 30 Goldman, D., Simmons, D., Swanson, I, Patrick, J. and Heinemann, S. (1986) Pmt. Natl.4cad. Sci. USA 83.4076-4080 31 Goldman, D., Deneris, E., Luyten, W., Kochhar, A., patrick, J_and Heinemann, S. (1987) Cell 48, -73 32 Blackman, J. G. (1970) Proc. Univ. Otc;o Med. Sch. 48.4-S 33 Axher* P.. Large, W. A. and Rang, H. P. (1979) 1. Pkusiol. &London) 295 X5;_-170 34 Gurney, A: M. and Rang, H. P. (1984) Br. 1. Phamtacol. 82. 225-242 3.5 Loring, R. H., Andrews, D., Lane, W. and Zigmond, R. E. (1986) Brain Res. 3%. ___,30-37 -- -. 36 Loring, R. H., Dahm, L. M. and Zigmond. R. E. (1985) Neuroscience14,645-
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37 Dryer, S. E. and Chiappinelli, V. A.
(1987) Neumscjence 20,9OS-910 38 Rang, H. I?. (1981) J. Pity&i. ILondon) 311,23-55 39 Colquhoun, D., Large, W. A. and Rang, H. P. (1977) J. Pkysiol. (Londorzf266,361-_3Y5
40 Sacchi, 0. and Petri, V. (1971) Pfliig. Arch. 239,207-219 41 Cull-Candy, S. G. and Mathie, A. (1986) Neurosci. Lett. 66,27%280 42 Mathie, A., Cull-Candy, S. G. and Calquhoun, D. Proc. R. Sot. London Ser. B (in PWS) 43 Derkach, V. A., North, R. A., Selyanko. A. A. and Skok, V. I. (1987) J. Physiof. 388,141-l% 44 Ogden, D. C., Gray, P. T. A., Colquhoun, D. and Rang, H. P. (1964) P&g. Arch. 400,44-S0 45 Bmhm, P. and Kullberg, R. (1987) Proc. Nat! Aced. Sci. USA 84,25SO-2554
Regulation of receptor function by protein phosphorylation Richard L. Huganir and Paul Greengard Most, if not all, membrane receptors appear to be regulated by protein phosphoqhtion. Richard Huganir and Paul Greengard describe the range of ~ec~~~s that are ~nvolued in these ~o~i~ca~on5 and propose II new scheme for clussificution of receptor regulation based upon the protein phosphorylation mechanisms involved. Many extracelhdar signals (first messengers), such as neurotransmittens, horrqones and growth factors, do not permeate the plasma membrane. instead, they interact with receptors at the cell surface, which in turn transduce the signals to the interior of the cell. It is known that the biological activities of membrane receptors are highly regulated and that this regulation plays a major role in modulating the sensitivity of cells to extracelhrlar signals. It is now clear that protein phosphorylation of receptors is the primary mechanism of regulation of receptor function. Major classes of membrane receptors Membrane receptors involved in signal transduction can be divided into three major classes which differ’both in their topological arrangements within the Richard Huganir is Assistant Professor and Paul Greengard is Professor end Head of the Laborutmy of Molecular and Cellular Neuroscience, The ~cke~ell~ Uniueni@. 2230 York Avenue. New York, NY 10021, USA. 8 1987.Eleevier Public&ions, Cambridge
plasma membrane, and in the molecular mechanisms by which they transduce signals. One class of receptors is the chemicallygated ion channels, and includes the nAChR, the GABA* receptor and the glycine receptor. These receptors are multimeric protein complexes which contain the receptor binding sites, as well as the ion channels which transduce the signals to the interior of the celi. A second class of receptors are linked to the guanine nucleotide binding proteins (G proteins) and includes the padrenergic receptor, the cyadrenergic receptor and the muscarinic receptor. These receptors are single polypeptide chains which contain the binding sites for the hormones; for these receptors to transduce the signal to the interior of the cell they must interact with the G proteins (see Gilman review in this issue). A third class of receptors is the growth factor receptors and includes the insulin receptor, the epidennal growth factor receptor and the platelet-delved growth factor receptor. These receptors
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1987 [Vol. 81
46 Gardner, P., Ogden, D. and Cofquhoun, D. (1984) Nature 309,160-142 47 Clarke, P. 6. S. (1986) in Nicotinic Acetylcholine Receptor Structure and Function (NATO ASI series H, Vol. 3) (Maelicke, A., ed.), pp. 345-357, Springer-Verlag 48 Martino-Barrows, A. M. and Kellar, K. J. (1987) Mol. Pkarmacol. 31,169-174 49 Kemp, G. and Morley, B. J. (1986) FEBS Left. 205,265-268 50 Whiting, P. and Lindstrom, J. (1987) Proc. Nat1 Acad. Sci. USA 84,595599 51 Lunt, G. (1986) Trends Neurosci. 9,341342 (see also foe. cit. 10,107) 52 Black, J. W., Jenkinson, D. H. and Gerskowitch, V. P. (eds) (1987) Perspectives on Receptor C~ass~~cfftion (Receptor Biochemistry and Methodology, Vol. 61 Alan R. Liss 53 Schuetze, 5. M. and Rote, L. W. (1987) Artnu. Rev. Neurosci. 10,403-457
have a single ~~smernbr~e domain. They contain the binding site for growth factors, as well as growth factor-regulated tyrosine kinase activity which is involved in transducing the signal into the interior of the cell. All three classes of membrane receptor are regulated by protein phosphorylation. The nicotinic ace~I~o~e receptor The nAChR is a neurotransmitter-dependent ion channel that causes the depolarization of the postsynaptic membrane in response to acetylcholine (see Changeux review pp. 459465.) The nicotinic receptor is a pentamerit complex which consists of four types of subunits LY (Mr 40 000), p (M, 50 000), y (M, 60 000) and 6 (M, 65000) in the stoichiometry of c&yG1 (see Fig. 1A). Isolated postsynaptic membranes enriched in the nicotinic receptor Coiit&fi dt leda. four distinct protein kinase(s): carp-dependent protein kinase; calcium/calmodulin-dependent protein kinase; protein kinase C; and a tyrosinespecific protein kinasez4. Three of these endogenous protein kinases phosphorylate the nAChR. CAMPdependent protein kinase rapidly and stoichiometrically phosphorylates serine residues on the y- and S-subunits of the recepto?. Protein kinase C rapidly and stoichiometricalljj phosphorylates a serine residue on the B-subunit and also causes a slower bat significant phosphorylation of a serine residue on the ar-subunit3. The endogenous tyrosine kinase(s), immunologically related to