Outstanding problems in acetylcholine receptor structure and regulation

3OO

12

13 14 15

16

M., Monmoto, Y,, Hirosc, I , Asax, M Inayama, S,, Nakanishi. S. and Numa. S. ( 19~;21 Nature ( L o n d o n ) 298, 245-249 Lamourou~, A., Fancon Biguel. N., Samolyk, D , PrivaI, A., Salomon, J. C.. Pujol, J, F. and Mallei, J. (1982) Proc. Natl A('ad .Sc.; U S.A. vt~ 3881-3885 Ling, N , Ying, S., Mm~ck, S and Guillemin. R. (1`479) Lift" Sci. 25, 1773-1780 Maxam. A. M. and Gilbert, W. (1977)Proc. Natl A ca d . Sci. U.S.A. 74, 56(>-564 Noda, M., Furutani, H., Takahashi. H., Toyosato, M., Hirose, T., lnayama, S.. Nakanishi, S. and Numa, S. ( 1982),Nature ( L o n d o n ) 295. 202-21)6 Nogata, 5;. Taira. H , Hall, S . Johnsrud, I ,

5;tzcuh. M.. Escodi, I . Boll, % .. Canlcll. K and Wetssmann, t" (198OJ ~'~lturt" ( L o n d o n ) 284, I7 ()kasama, H. and Berg. P. (1'482) MoL ("ell Bi~,/ 2. 161-170 IS Parne~. J R.. Velan. B., Felsenleld, A.. Ramanathan, L.. Ferrim, U . Appella, E. and Seldman, J. G. (19811Pro(' Natl Acad. S(i. L'.."; .t. 78. 2253-2257 ~'4 Ruddle. F. H (ItJ8!)X/ature ( L o n d o n ) 284. I I5~i20 20 Sanger, F.. Nicklen, S. and Coulson. A. R. ~ 1977) t'r~*c. Natl Acad. Sci. U.S..4. 74. 5463-5467 21 Southern. E. M. (1975)). MoL Biol. 9 8 . 5 0 3 - 5 1 7 22 Sutclifl~'. J. G.. Milner, R. L, Bloom. F. E. and

Outstanding problems in acetyicholine receptor structure and regulation Jim Patrick and Steve Heinemann Research on acetylcholine receptor addresses two general and very important questions. Acetylcholine receptor is a ligand-gated ion channel and one goal is to determine precisely how binding o f acetylcholine to this transmembrane protein results in the production of a n ion channel. Acetylcholine receptor is also a muscle protein with its properties dependent upon innervation. In this case, the goal is to determine how the innervating neuron determines which o f several properties the receptor will possess. Understanding how acetylcholine receptor functions will very likely be o f value in understanding how the many other neurotransmitter receptors" function. Knowledge o f how innervation determines receptor properties will probably contribute both to our understanding o f how the nerve controls other muscle properties and to our appreciation o f synaptogenesis in general. Acetyleholine receptor function and structure The early studies of acetylcholine receptor were dependent upon the only assay available - the ability of receptor to depolarize the post-synaptic membrane in the presence of a ligand. These changes in membrane potential allowed study of the ionic composition of the membrane current, the spectrum of ligands which could induce and block receptor activation, and the various states of activation and inactivation of the receptor. The assay was indirect, however, since it measured voltage changes, consequences of current, rather than the opening and closing of the ion channels. Application of voltage clamp procedures permitted measurement of the current, rather than the voltage, and thus moved a step closer to the event of interest, the interaction of acetylcholine and receptor to generate an open ion channel. These studies were rapidly followed by experiments designed to determine the rate and extent to which individual receptor molecules could generate ionic current. A statistical analysis of the noise

associated with the tigand-dependent opening and closing of receptor channels made it possible to calculate the conductance of a single ion channel and the rate and voltage dependence of channel closing. The actual shape of the current pulse associated with opening and closing a receptor channel was not obtainable through analysis of noise. This information required procedures which monitor the opening and closing of individual receptor molecules. The ability to make surface electrodes which form very high resistance seals with the membrane allows measurement of the picoampere currents which flow through single ion channels. This patch clamp technique now offers unprecedented access to the functioning not only of acetylchotine receptors but also of a wide variety of ligand- and voltage,gated ion channels. The techniques are now well in hand for a thorough analysis of acetylcholine receptor functions, ~7.~s. For years, structural work on the acetylcholine receptor lagged far behind studies of function because techniques for purification and structural analysis were not avail-

t.erner. R..~. / % . . . \ a t /

,had

~,

, ~3

press) 23 Thomas, P (1980) }'r:,t, %,zl/ .ll~d ;., ; l ~ 1 77, 5201-5205 24 Williams, J G ¢ )981} i11 (;em.tw tneittectm~t (Williamson, R cal.). Vol } Academic Pro.... New York 25 Wu. R. (ed.) (1'979) ~dethods ill I~nz~mt~[oq.~ , Vol. 68, R e c m n h i n a n t D N A , -\cademic Pres. New York

Robert J. Milner L~ at the A . V. l)avts ( enter Jot Behavioral Neurobiotogy, The Salk lnstitau', P O. B o x 85800, San l)iego, ¢ A 9 2 1 3 & ~'.'~

able. From the beginning the problem was how to work with a molecule identifiable only by its ability to depolarize the cell in which it resides. Early biochemical studies used the neurotoxin d-tubocurarine, which blocks receptor activation, to label the receptor molecule in cell extracts. The relative low affinity of d-tubocurarine for receptor made it difficult to detect specific binding in the presence of the non-specific background binding. A significant advance came with the discovery that snakes of the family Elapidae produce a vmrom containing a polypeptide toxin that blocks activation of acetylcholine receptors in most skeletal muscle. These polypeptide o~-neurotoxins, which were shown to bind with very high affinity and exquisite specificity to the acetylcholine receptors, made acetylcholine receptors available to biochemical techniques. The fact that the ~e-neurotoxins could be labeled with isotope or fluorescent chromophores made it possible to qaantitate and visualize receptors in the cell surface without recourse to function. The fact that toxins would bind specifically to rcceptor that had been solubilized from the membrane made possiblc its detection in solution and led quickly tu its purification:'. '~' Purification of acetylcholine receptor flom mammalian muscle is difficult since the synapse constitutes a small fraction of the muscle and the total amount of receptor available is very small. This problem was overcome by using the electric organs of various rays and fishes in which modified muscle cells are used to produce strong electric fields. In the ray (Torpedo sp. ) the field is generated by activating acetylcholine receptors present in very high density in the electroplax membrane. Large amounts of acetylcholine receptor can be purified from these tissues. Consequentls.. the best structural data available Ior the receptor are for that purified from Torpedo electric organ 5.10. Receptor from this source is thought to be composed of tour different polypeptide chains, ~, /3, y and ~, of 4t), 50, 60 and 65 kD respectively. The receptor

7INS - September 1982 is a pentamer of the composition (a)~/3yS. The sequences of the first 50---60 amino terminal amino acids of each polypeptide have been determine@% The interesting result of this first study of receptor primary structure is that there is considerable homology between these polypeptides at their N-terminal ends, suggesting they have a common ancestor. Clearly, elucidating the complete amino-acid sequence for each polypeptide will provide valuable information not only about the ancestral relationships between the polypeptides but about the relationships that exist between these polypeptides within the membrane. Membranes containing high densities of receptor can be shadowed or negatively stained and the receptor visualized in the electron microscojc~e. Receptor appears as a disc of about 90A in diameter with a low density region or pit in the center. Other experiments indicate that the receptor peptides span the membrane extending about 55A on the extracellular side and about 15A on the cytoplasmic side n. The polypepddes appear to be arranged in a circle about the central pit. From the stoichiometry of the polypeptides, known disulfide bonding between polypeptides, and specific toxin and antibody markers it is possible to inler an order of polypeptides around the circle TM. A complete description of the structure of receptor in the membrane requires procedures to visualize known sequences in each subunit. Knowing the cumplete amino acid sequence of each subunit will greatly facilitate this process. The primar~ structure for Torpedo receptor can probably be obtained most readily from the sequence of the DNA which codes for each receptor polypeptide. This work has begun with the recent isolation of a eDNA chine coding for the y or 60 Kd subunit of 7brpedo acetylcholine mceptorL This clone was obtained from a eDNA library prepared from mRNA isolated from Torpedo califi~rnica electric organ. Identification of the clone as coding for the y subunit was achieved by sequence analysis. The clone contains sequences appropriate lot a typical signal peptide followed by a sequence which matches the amino terminal amino acid sequence of the 3/ subunit. This approach will lead to the determination of the complete amino-.acid sequence of each mature receptor polypeptide as well as the sequence of the leader peptide present in their biosynthetic precursors. Potential carbohydrate attachment sites can be identified in the amino-acid sequence, but precise allocation and identification of the carbohydrate clearly requires biochemical analysis of the mature protein. Likewise, markers lor specific por-

301 degraded much more slowly. Just how these classes of receptors are generated and how the nerve regulates their re]ative abundance is not known. It is clear, however. that the electrical activity induced in the muscle by the nerve plays a role in regulatir g the appearance of the rapidly-turningc ,,er class of receptors';. There are antigenic differences between ;eceptors on the muscle fibeff'. Individuals with myasthenia gravis produce antibodies against their own acetylcholine receptors Regulation o f acetylcholine r e c e p t o r and some of these antibodies recognize rat properties acetylcholine receptor. Within the antibody While purification of large quantities of population that recognizes rat receptor there acetylcholine receptor from mammalian are antibody specificities that do not recogmuscle is difficult, isotopically labeled nize acetylcholine receptor lound at the neurotoxins have made possible studies of synapse but do recognize receptor that many aspects of mammalian receptors. The appears after the muscle is denervated. ability to detect temptomole quantities of Furthermore, when acetylcholine receptors receptor on muscle cells have also made were analysed by isoelectric lbcusing the possible detailed studies both of receptor receptor found at the synapse locused synthesis during development and of about 0.15 pH units lower than the receptor metabolism of the receptor molecule. found tollowing denervation 4. Therelore, These studies revealed some very interest- in addition to their distribution and ing properties of receptor. As expected metabolic stability, receptors can be distinfrom studies relying upon electrical mea.,,- guished on the basis of the antigenic deterurements, the distribution of receptor on the minants they carry and their position in an innervated muscle surface is non-uniform. isoelectric focusing gradient. Receptors are clustered in the post-synaptic Acetylcholine receptors at the rat membrane, specifically' on the tips of the neuromuscular junction function differpostjunctional folds in the post-synaptic ently from those that appear following membrane. In the absence of innervation, denervation '4. Receptors can be characterhowever, the receptors are generally distri- ized by how long the ion channel stays open buted diffusely over the muscle membrane. in the presence of ligand. Receptors that are The way in which the nerve causes the diffusely distributed over the surface of the receptor clusters to be localized in the muscle stay ()pen, on average, twice as long immediate post-synaptic membrane is an as those that are localized at the neuromusimportant question in synaptogenesis. cular junction. Acetylcholine receptor is synthesized, The results described above demonstrate inserted into the membrane, and then that there are two classes of acetylcholine removed from the membrane and receptors on skeletal muscle - those that are degraded ''~. This metabolic pathway was junctional, localized in the post-synaptic demonstrated by examination of the membrane, and those that appear extrajuncappearance and loss of neurotoxin binding tionally following denervation. These sites, and the co-degradation of receptor receptors difler in their distribution, their and bound isotopically labeled toxin. The metabolism, their antigenic properties, and fact that toxin bound to receptor and did not in their open-times. The nerve appears to dissociate noticeably allowed measurement regulate this process since the presence or of the metabolic hall-life of the receptor. absence of the tv, o classes is dependent These studies revealed an important fact upon the presence of functional innervaabout acetylcholine receptors. Receptors lion, If one knew the basis lor the diflerlocalized in the post-synaptic membrane ences between these receptors and the are metabolized much more slowly than mechanism that controls their relative those that are distributed diffusely over the abundance one would know how the nerve surface of a non-innervated muscleL determines at least one specific muscle Receptors on non-innervated muscle have a phenotype. Since the receptor molecules metabolic half-life of about 17 h. Alter have different properties, the first question innervation, the receptors constrained to the is "what is the molecular basis of the difierpost-synaptic membrane have a metabolic ences?' A number of models are possible. half-life of about 10 days. If the muscle is There may be a mechanism under neural denervated, the receptors that appear over control that modifies receptors, changing the surface of the muscle have a metabolic them from onc type to another. Phosphoryhalf-life of about 17 h while those that were lation of one or more receptor subunits found at the endplate continue to be might be such a mechanism7'". Allernations of each polypeptide can be produced by synthesizing short peptides corresponding to specific sequences in the mature polypeptide. Antibodies made against these short peptides will be invaluable for structural, functional, and anatomical studies. In addition, they may help define the nature of the immune response in the disease myasthenia gravis which is known to involve an immunological attack on acetylcholine receptor ~, %

I'IAA' -..Sc/mmi~cr ! 9 & "

302 lively, there may be a mechanism under neural control that causes the synthesis of one kind of receptor in preference to another. Toxins and biophysical techniques have been the tools that revealed the exi~ tence of two classes of receptors. The techiques of recombinant DNA are particularly well suited to revealing both the basis of the differences between the two classes of muscle acetylcholine receptors, and the ways in which the nerve might generate the difference. The amino-acid sequences of junctional and extrajunctional acetylcholine receptor can be obtained and compared using eDNA clones of the two receptor types. If the amino-acid sequences are different, then the differences between the two receptor classes must be a consequence of some difference in gene expression. In this case molecular cloning techniques make it possible to determine the genomic organization of acetylcholine receptor genes, reveal differences in their expression and, eventually, help determine how the nerve controls expression of receptor coding sequences. The emphasis is not on the receptor m o l e c u l e p e r se but on the ways in which the nerve controls receptor properties. In this case, acetylcboline receptor is rapidly becoming a manageable marker of muscle phenotype and therefore a way in which to study how the trophic interaction of nerve and muscle regulates muscle development.

Other types of aeetylcholine receptors The acetylcholine receptor found on skeletal muscle is called a nicotinic acetylcholine receptor because it is activated by the ligand nicotine. Nicotinic acetylcholine receptors are also found on nerve cells where they differ in several ways from those found on muscle. Some ligands that activate nicotinic receptors on muscle block nicotinic receptors on nerve, and e~-neurotoxins that block activation of nicotinic receptors on muscle fail to block activation of nicotinic receptors on nerve. These two kinds of receptors do however have a common pharmacology and control ion channels with similar characteristics, Little is known about the structure of the nicotinic acetylcholine receptors on nerve; nor is it known whether, in mammals, innervation plays a role in regulating the properties of this receptor, although nerve does regulate the distribution of acetylcholine receptor on nerve cells in froglL A second general group of acetylcholine receptors are characterized as muscarinic:L These receptors are not activated by nicotine but are activated by the ligand muscarine. Muscarinic acetylcholine receptors are found in smooth muscle, ganglionic fibers, some chromaffin cells, and in the

CNS. In contrast to the nicotinic receptors. which all appear to work by changing membrane conductance, activation of m u v carinic receptors is in some cases coupled to nucleotide cyclasc systems. The m u ~ carinic receptors are tess easily studied, in part because there is no analogue to the electric organ to provide quantities of mattrial and in part because the reagents available lor labeling muscarinic receptors arc not as convenient as the snake toxins. Consequently, the study of these receptors has been more difficuh,

Prospectus All the acetylcholine receptors together may form a family of related proteins. They share such attributes as the ability to bind acetylcholine and acetylcholine analogues and a transmembrane location; many open ion channels when activated. In general, they serve as neurotransmitter receptors and the innervating neuron can regulate at least the abundance of both nicotinic and muscarinic receptors. If these similarities exist because acetylcboline receptors have a common ancestor we might find aminoacid sequences conserved between all or at least some of these receptors. If this were true. antibodies raised against one acetylcholine receptor might be expected to recognize another type of acetylchotine receptor. This will not be the case if conserved sequences happen not to be the ones that induce antibody production. However, synthetic peptides corresponding to defined regions of the protein might induce useful antibodies. An alternative approach is to use sequences of DNA which code for conserved amino-acid sequences. A portion of the eDNA lot muscle nicotinic receptor may hybridize to genes for other receptors such as the nerve nicotinic receptor, various muscarinic receptors and perhaps to as yet unidentified receptors. Similarly, the portion of the receptor that forms the ion channel may have considerable homology with ion channels regulated by other neurotransmitters. Complete homology of sequence is not required since the conditions for D N A - D N A hybridizations can be adjusted to retain hybrid molecules with as much as 35% difference in nucleotide sequence. The fortunate situatkm in which one could find nucleotide sequence homology between classes of acetylcholine receptor would provide a very powerful approach to the study of this group of proteins. If a related gene can be identified by hybridization the sequence of the protein for which it codes can be deduced. As noted above, peptides with sequences corresponding to interesting regions of the protein can be synthesized and used to generate antibodies

which provide lools for identification of the protein in viw). Our understanding of acetylchohnc receptor structure, function and regulation has advanced in major steps as new ideas and techniques were used, The technology of recombinant DNA is available lo provide a new step which will enlarge our unde> standing of the nicotinic receptor ~m musc]e and which may make a wide variety of dill. ferent kinds of acetylcholine receptors available lot' study.

Reading list I Ballivet, M.. Patrick. J.. Lee. J. and Hemematm. S. Pro< Natl Acad. Sci. U.S.,4. (in pres,,) 2 Berg. D. K, and Hail. Z, W. 119751J. PhysioL (Londont 252,771 3 Birdsall.N, J M.. Hulme. E. C.. Hammer. R. and Stockton, I. S, (1980) l~vyehopharmaeology attd Biochemist O' of Neurotransmitter

Receptors

(Yamamttra, H. I., Olsen. R. and Usdin. E,. eels), pp. 07- I(X) 4 Brockes,J. P. and Hall. Z. W. ( 19751Biochemtstr~ 14. 2100 5 Changeux. J. P, 119811ttarvQ' Lecture Serie~ 75. AcademicPress 6 Fambrough. D M. (19791 Phy~ud. Rev. 3, 756-824 7 Gordon, A, S. Davis, C. O., Millay. D. and Diamond. t (l~77)Proc. Natl Acad. S'ci. U.S,A. 74. 263-267 8 Hamill. O. P.. Many. A., Neher, E . Sakmann. B, and Sigworth, I- J. 11981)Pfluegers' Arch. 391. 85-1(~) 9 Hohzman. E.. Vqise.D,. Wall, J. and Karlin, A ( 1982)Prow. Nag A~'ad. Sci, U.S.A. 79, 310-3 t 4 10 Karlin. A. (I980) The (ell ~'urface and Neuronal Function tCotman. (', Poste. G. and Nicolson. G. L,. eds), pp 191-260. Elsevier/North-HollandBiomedicalPress 11 Kis/ler. J., Stroud, R. M., Klymkowsky,M. W.. Lalanceue, R and Fairclough. R H 09821 Biophys, J.

12 Kaffier. S. W . Dennis, M. J. and Harris, A..L (19711 Proc. R Soc. London, B Ser. 177 555-51~3 13 Lindslrom. J. and Engel, A. 11981 ~ Receptor Regulation (Lefkowitz,R. J., ed.). pp. 163-214 14 Neher, E and Sakmann, B. 11976),1. Phwiof (London) 258. 705 15 Patrick, J. and Berman, P. W. (19811) The (ell Surface and Neuronal Function (Cotman, C., Poste. G. and Nicolson, G L., eds). 157-190, Elsevier/North-HollandBiomedicalPress 16 Raftery, M. A.. Hunakpiller, M. W., Strader. C. D. and Htw,d, L. E. 119801 Science 208, 1454-1457 17 Steinbach. J. H. 119801 The (_ell Surface and Neuronal Function (Cotman. C. W. Poste. G and Nicolson, G. L., eds), Vol. 6, Elsevier/North-HoUandBiomedicalPress 18 Stevens, ('. F. (1980)Annu. Rex,. Physiol. 42, 643-652 19 Teichberg. V. t and Changeux, J. P. 11977) FEBS Lett. 74, 7 t-76 20 Vincent,A. (1980)Physiol. Rev. 3,756-82,4 21 Wemberg, C. B. and Hall, Z. W (1970) Pr,c.c. Natl Acad. Sci. U.S.A. 76, 504 Jim t~atrick and Steve Heinemann are at the Molecular Neurobiology Laboratory. The Salk Institute, P.O. Box 85800. San Diego, CA 92138. U.S.A.