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THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. DATE: Dec. 6, 1988 MEMORANDUM: TO: Dr. James F. Glenn, and Staff FROM: Dr. D.H. Ford RE: Site visit with Dr. J. Lindstrom, The Salk Institute, San Diego, CA, November 16, 1988 Grant No. 2107R1 entitled "Neuronal nicotinic receptor structure" Site visitors: D. Ford and D. Stone Goal: To determine the structure of the nicotinic acetylcholine receptor in muscle, adrenal medullary tumor cells and on cells of the central nervous system and to compare nACh receptors from the brains of various species. Results: This has been a very active group, as evidenced by their:,_ recent publications (list appended). One not listed is entitled "Expression of the four subunits of the Torpedo californica Nicotinic Acetylcholine Receptor in Saccharomvices cervisae " a yeast. To summarize briefly, it appears that there are two types of peptide in the nACh receptor. Those which are structural subunits (beta, gamma and delta) and one which binds ACh, the alpha unit. If one examines the amino acid sequence of the alpha subunit from muscle receptors of mouse, calf, chicken., xenopus and from a human cerebellar medullablastoma cell, he reports a marked degree of homology amongst these six types. In all of them ACh binds to a site near two disulfide-linked cystines, which are extra cellular. While there are many homologus regions among the structural peptides from these same six receptors, they are much less impressive. The receptor binding site is followed by three extremely homologus regions (MI,2 and 3), which are located within the cell membrane. By immunoaffinity purification, the ACh binding subunit of the eel electric organ and muscle is 40kD, while the ACh binding unit of chick brain is 59kD; in rat brain, 75kD; in man is 79kD and in the calf is 75kD. The structural units for chick, rat, man and calf are all about 50kD. If one examines the stoichiometry of the various subunits, it is apparent that in the Torpedo electric organ, there are 2 alpha units and one each of the beta, gamma and delta units. For chick, rat and bovine brain there appear to be 2 alpha units for 2 beta units, for the most likely configuration.

2 In a recent study of chick retina, they have observed, by the use of antibodies that there is a marked accumulation of receptor protein in the ganglion and amacrine cells of the retina. Immunocytochemical studies have also revealed a dense accumulation of nACh receptors in the rat cerebral and cerebellar cortex, hippocampus, striatum and thalamus. Similar accumulations were noted in the frog optic tectum and along the terminals of the optic tract. The studies which the Lindstrom group have completed with yeast indicate that the cDNAs from the electric eel are capable of inducing the synthesis of the all four peptide types, but the yeast cell is unable to assemble them to form a receptor. By comparison, the frog oocyte is able to accomplish this. What now appears evident from the work of Lindstrom, and others, is that all the various ACh receptor message subtypes are part of an extended gene family, and that this family includes other ligand-gated channels, such as the receptors for GABA, glycine, 5-HT, Sub P and neurokinin K. The last three have all been shown to open ion channels and to be Ca dependent. The subsequent response of these three then depends on activation of specific G proteins. The question which now arises is: with ion-gated channels, is there a specific extramembrane region (like the cystine-rich area for the nACh receptor) which recognizes a specific ligand + an intramembranous segment in which there appears to be many homologies and then finally a specific intracytoplasmic portion which activates a specific second messenger sequence? This is extremely interesting work which has already extended our understanding of the nACh receptor. The theory which derives from his work that the intramembranous portion of the receptor which forms the ion channel may represent a highly conserved region which may be more or less common to many types of receptor is most exciting. Question: Do pre- and postsynaptic receptors utilize the same second messengers? If one is ever to understand the various effects induced by nicotine within the CNS, a thorough understanding of this receptor is virtually mandatory. A colleague of Dr. Lindstrom's, Dr. M. Montal spoke with us about the work he is undertaking. His immediate goal is to determine how the receptor protein functions. That is, which parts form the pore structure, channel lining and which segments confer gating specificity to recognize a specific ligand; how is the sensor domain coupled to the channel domain, etc. He has sent in a letter of inquiry and will be sending in an application for consideration at the April 1989 SAB meeting. Don Ford DF/ff