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THE COUNCIL FOR TOBACCO RESI:ARCH-U.S.A., INC. August 15, 1985 TO: Dr. S. C. Sommers and Staff FROM: D. H. Ford and D. Stone RE: Site visit with Dr. L. Hal~kat the Albert Einstein College of Medicine, Bronx, NY, May 24, 1985 Grant No. 1126BR2 entitled "Genetic Differences in Nicotine Sensitivity in Drosophila Melanogaster Strains" GOAL To characterize the nature of the nicotine binding sites in Drosophila in normal wild type flies as compared to nicotine resistant mutants. PROGRESS During the past funding period Dr. Hall has concentrated on characterizing the 3H-nicotine binding site on neuronal membranes extracted from Canton-S-wild type flies, using a centrifugal assay. Initial studies on the binding of tritiated nicotine:,indicated that the total number of binding sites (Bmax) was 2.18 + 0.59 pmole/mg of protein, which is similar to the Bmax obtained with 125I-4-bungarotoxin in this type of prep- aration (3.00 + 0.55 pmole/mg protein). Scatchard analysis of the binding at different concentrations of nicotine (Figs. 1 and 2) suggests that the curves obtained reflect cooperativity in binding; that is: the nicotine is binding to multiple classes of binding sites. This result has been repeated 12 times. (A straight line plot would have suggested that the nictoine binding was to only one type of receptor, while the curvilinear plot obtained suggests cooperativity of binding between more than one type of receptor.) This is particularly interesting in that it implies that nicotine receptors in the fruit fly may be comparabale to those in the mammal and that this type of cooperativity in binding sites by a single transmitter (i.e., acetylcholine binding to nicotinic type receptors) has enjoyed a long phylogenic history. Dr. Hall is now investigating new methods of computer analysis for evaluating these curvilinear Scatchard plots produced by the interactions between nicotine-and its binding sites. .4. Recently Dr. Hall exhausted her supply of 3H-nicotine obtained from New England Nuclear and found to her consternation that the next batch obtained was contaminated (an experience duplicated by many others). She is presently testing a new batch to be sure it is uncontaminated before proceeding with experiments. Once this is accomplished she plans to determine if preincubation of the receptor protein with unlabelled nicotine will alter the shape of the saturation curve. This could be expected if the nicotine itself induces a change in the state of the receptor. She also plans to compare the curves she obtains now using DL-nicotine with what'is obtained using only the L isomer (obtained from Amersham in England). All these comparisons will be done in nicotine resistant mutants as well as in wild type control flies.

Site Visit May 24, 1985 Dr. L. Hall (#1126BR2) Page 2 OTHER STUDIES A comparison of age on nicotine binding has been initiated. Early results suggest that age has little or no effect on the affinity of binding. Dr. Hall has also started to culture isolated neurons (with a few glia), which are obtained from Drosophila larvae, to determine the effect of nicotine on the growth of neuronal processes. Neurons in these cultures grow and establish synaptic connections and have been shown to be excitable. She plans to use this model to study ion channel activity, using a "patch clamp" technique. Finally, once the binding studies described above are completed, Dr. Hall intends to refocus a part of her efforts on cloning the genes affecting nicotine resistance. Question: Are the nicotine resistant receptors the result of different gene products than normal receptors or are the two receptor types intraconvertible with each other? COMMENT This continues to be an interesting investigation on the characteristics of the nACh receptor and how it is influenced by nicotine. As has been observed in mammals, the fruit fly now appears to possess at least two types of nACh receptor under normal conditions as well as being modifiable by an alteration in protein organization to a mutant receptor, which is nicotine resistant. This presumably is induced by an effect at the genomic level. Thus, the fruit fly continues to be a useful model for investigating the interactions between nicotine and the nACh receptor. The introduction of the cultured neuron model should make this an even more interesting program. DHF/DS

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