Rhesus monkey α7 nicotinic acetylcholine receptors: Comparisons to human α7 receptors expressed in Xenopus oocytes

Abstract

An α7 nicotinic acetylcholine receptor sequence was cloned from Rhesus monkey (Macaca mulatta). This clone differs from the mature human α7 nicotinic acetylcholine receptor in only four amino acids, two of which are in the extracellular domain. The monkey α7 nicotinic receptor was characterized in regard to its functional responses to acetylcholine, choline, cytisine, and the experimental α7-selective agonists 4OH-GTS-21, TC-1698, and AR-R17779. For all of these agonists, the EC₅₀ for activation of monkey receptors was uniformly higher than for human receptors. In contrast, the potencies of mecamylamine and MLA for inhibiting monkey and human α7 were comparable. Acetylcholine and 4OH-GTS-21 were used to probe the significance of the single point differences in the extracellular domain. Mutants with the two different amino acids in the extracellular domain of the monkey receptor changed to the corresponding sequence of the human receptor had responses to these agonists that were not significantly different in EC₅₀ from wild-type human α7 nicotinic receptors. Monkey α7 nicotinic receptors have a serine at residue 171, while the human receptors have an asparagine at this site. Monkey S171N mutants were more like human α7 nicotinic receptors, while mutations at the other site (K186R) had relatively little effect. These experiments point toward the basic utility of the monkey receptor as a model for the human α7 nicotinic receptor, albeit with the caveat that these receptors will vary in their agonist concentration dependency. They also point to the potential importance of a newly identified sequence element for modeling the specific amino acids involved with receptor activation.

Introduction

The α-bungarotoxin sensitive α7-type nicotinic acetylcholine receptor is expressed throughout the brain and also in the peripheral nervous system and some peripheral tissues (Sharma and Vijayaraghavan, 2002). In the brain, α7 nicotinic acetylcholine receptor are located in high concentrations in the hippocampus, neocortex, and hypothalamus as seen by binding sites (Clarke et al., 1985). The use of nicotinic agonists has improved delay matching in primates (Terry et al., 2002), eye blink memory in rabbits, and spatial-memory related behavior in rats, as well as social memory relationships in rats (Arendash et al., 1995a, Arendash et al., 1995b, Meyer et al., 1994, Van Kampen et al., 2004). Some mutations and/or splice variants of the α7 gene have been linked to a decrease in hippocampal auditory gating, which is a symptom of some schizophrenics and approximately 50% of their family members (Freedman et al., 1994, Freedman et al., 2000). This may be due to the roles played by α7 nicotinic acetylcholine receptor in the activation of GABAergic inhibitory interneurons in hippocampus (Adler et al., 1998, Frazier et al., 2003). The α7 nicotinic receptor may also be involved with the etiology and/or possible treatment of other conditions such as Alzheimer's Disease and Down's Syndrome. Nicotinic receptor agonists have been shown to improve memory and are neuroprotective. Moreover, α7 has been found to coprecipitate with the Aβ_1-42 within the histopathological amyloid beta plaques (Wang et al., 2000) and the functional interactions between Aβ_1-42 and α7 nicotinic acetylcholine receptor (Liu et al., 2001) further support α7 nicotinic acetylcholine receptor as a therapeutic target for Alzheimer's Disease.

The adverse side effects of nicotine or other non-selective cholinergic agonists have promoted the development of more selective α7 agonists for therapeutics. GTS-21 (2,4-dimethoxybenzylidene anabaseine or DMBX), one such selective agonist was tested in phase 1 clinical trials and was found to have no adverse side effects and to increase cognitive functioning in healthy subjects (Kitagawa et al., 2003). With the identification of α7 nicotinic receptors as potential therapeutic targets has also come the need to develop animal models for the testing of novel therapeutic agents. While rodent models are most commonly used, there are numerous pharmacological differences between rat and human α7 nicotinic receptors (Papke and Papke, 2002). Monkey models have the intrinsic advantage of being amenable to more complex behavioral testing than rodents and therefore may have special usefulness for evaluating potential drugs for human therapeutics. We report the cloning and functional characterization of a Rhesus monkey (Macaca mulatta) α7 nicotinic acetylcholine receptor (mkα7) in regard to its responses to a series of nicotinic agonists including acetylcholine (acetylcholine), choline, and cytisine, as well as the α7-selective agonists 4OH-GTS-21 (4-hydroxy 2-methoxybenzylidene anabaseine), TC-1698 (2-(3-pyridyl)-1-azabicyclo[3.2.2]nonane) and AR-R17779 ((−)-spiro[1-azabicyclo[2.2.2]octane-3′,5′-oxazolidin-2′-one) (Marrero et al., 2003, Meyer et al., 1998, Papke et al., 2004). These agonists had lower EC₅₀s for activating human α7 (hα7) nicotinic receptors than for monkey α7 (mkα7) nicotinic receptors. Only two amino acids differ between the mkα7 and hα7 sequence in the extracellular domain. Mutations were made of mkα7 sequence to the amino acids present in hα7, and the resulting mutants were tested to determine if changing either or both of the amino acids shifted the concentration–response relationships towards that of hα7.