1 Pharmacological studies of a7 nicotinic acetylcholine receptors are confounded by the fact that rapid desensitization to high agonist concentration causes a7 peak responses to occur well in advance of complete solution exchange. For this reason, peak currents are an invalid measure of response to applied agonist concentrations. We show that results comparable to those that have been corrected for instantaneous concentration are obtained if net charge is used as the measure of receptor response. 2 Dose response curves obtained with these methods indicate that a7 receptors are approximately 10 fold more sensitive to agonist than previously reported. The agonists, ACh, choline, cytisine, GTS-21, 4OH-GTS-21 and 4-MeO-CA have the same rank order potency for both human and rat receptors: 4-MeO-CA 4 4OH-GTS-21 4 GTS-21 4 cytisine 4 ACh 4 choline. However, di erences in e cacy exist between rat and human receptors. GTS-21 is more e cacious for rat than human a7 receptors and cytisine more e cacious for human than rat a7 receptors. 3 Choline is the least potent agonist for both human and rat a7, with a potency approximately 10 fold lower than that of ACh. While the EC50 for the activation of a7 receptors by choline (400 ± 500 mM) is outside the normal physiological range (10 ± 100 mM), choline can nonetheless produce detectable levels of channel activation in the physiological concentration range. Since these concentrations are relatively non-desensitizing, the contribution of choline-activated a7 receptor current may play a signi®cant role in the regulation of calcium homeostasis in a7-expressing neurons.

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The ␣7 nAChR-selective partial agonist 3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) is more efficacious and potent for rat receptors than for human ␣7 receptors. Four single amino acid differences exist between human and rat ␣7 in the agonist binding site, two in the C loop, and one each in the E and F loops. Reciprocal mutations were made in these three domains and evaluated in Xenopus laevis oocytes. Mutations in the C and F loops significantly increased the efficacy of GTS-21 for the human receptor mutants but not to the level of the wild-type, and reciprocal mutations in rat ␣7 did not decrease responses to GTS-21. Whereas mutations in the E loop alone were without effect, the E-and F-loop mutations together increased GTS-21 efficacy and potency for human receptors, but the EF mutations in the rat receptors decreased the GTS-21 potency without changing the efficacy. The only mutants that showed a full reversal of the efficacy differences between human and rat ␣7 contained complete exchange of all four sites in the C, E, and F loops or just the sites in the C and F loops. However, the reversal of the potency ratio seen with the EF mutants was not evident in the CEF mutants. Our data therefore indicate that the pharmacological differences between rat and human ␣7 receptors are caused by reciprocal differences in sites within and around the binding site. Specific features in the agonist molecule itself are also identified that interact with these structural features of the receptor agonist binding site.A crucial assumption for the translation of preclinical research from animal studies to human therapeutics is that receptor pharmacology will be consistent between species. That is, drugs shown to be useful based on their ability to work in animal (rodent) models would also have similar activity on human forms of the receptors. The neuronal ␣7-type nicotinic acetylcholine receptor (nAChR) has been identified as a potential target for the treatment of Alzheimer's disease (Lindstrom, 1997), and 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21; also called DMXBA), which selectively targets this receptor, has been shown to improve learning and memory in animal models of cholinergic hypofunction (Kem, 2000). This ␣7-selective partial agonist has also been shown to prevent the death of differentiated PC-12 cells that occurs after nerve growth factor removal and the death of cultured primary neurons that occurs after high levels of NMDA receptor activation (Martin et al., 1994;Shimohama et al., 1998). It is interesting that although GTS-21 was able to protect PC-12 cells from the cytotoxic effects of amyloid peptide exposure, it was not able to protect human-derived SK-N-SH cells from the same cytotoxic stress, although the GTS-21 4-hydroxy metabolite, 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4-OH-GTS-21), was cytoprotective in the same assay (Meyer et al., 1998a). A likely explanation for these observed differences in cytoprotective activity came from the observation that GTS-21 was far less efficacious ...

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