Multiple components in the agonist concentration–response relationships of neuronal nicotinic acetylcholine receptors
Introduction
Recently there has been much interest in developing agonists of neuronal nicotinic acetylcholine (ACh) receptors as therapeutic agents (Gopalakrishnan and Donnelly-Roberts, 1998). Knowledge of the agonist concentration–response relationships for different subunit combinations gives an indication of the subtype selectivity of the agents and how a native receptor of similar subunit composition may respond in vivo in disease states such as epilepsy (e.g. Steinlein et al., 1997). These studies can also reveal whether receptors are likely to be affected by low levels of agonist sometimes found during the abuse of drugs such as nicotine, and have been used to identify the presence of supplementary receptor subunits in receptor combinations (Ramirez-Latorre et al., 1996). It is also important to know the shape of such curves for studies of receptor modulation, since the amount of change induced by a modulator can be different at different points on the concentration–response curve (Lin et al., 1992). In the past, concentration–response relationships obtained from studies in oocytes have been analysed assuming there is a single component in the concentration response. In order to assess the validity of this assumption, and to predict how different neuronal nicotinic receptor subtypes may respond over a full-range of agonist concentrations, we examined the concentration–response characteristics of several subunit combinations in considerable detail.
Section snippets
Functional expression in Xenopus oocytes
Diguanosine-triphosphate capped RNA and defolliculated oocytes were prepared for injection as described previously (Covernton and Connolly, 1997). Up to 20 ng of RNA encoding subunits of rat nicotinic ACh receptors (AChRs) were injected into the oocytes in a ratio of ≈1:1.5 (α:β).
Drug application protocol
Two electrode voltage clamp recordings (VH=−60 mV) were obtained as described previously, (Covernton and Connolly, 1997). Pipette solutions had the following composition: current pipette CsF 0.25 M, CsCl 0.25 M, 100 mM
Typical agonist responses of nicotinic receptors
Fig. 1a shows an example of the high concentration ‘one-shot’ responses (α4–1β2) used to construct the curves, while Fig. 1b, c, d, and e show representative responses from the other receptor combinations tested. It is interesting to note that despite the replacement of Ca2+ by Ba2+ in the recording solutions, the rate of desensitisation can vary widely between the different combinations at similar concentrations of agonist.
Acetylcholine concentration–response relationship of the α4–1β2 combination
Fig. 2a shows the concentration–response relationship for the action of
Comparison with previous studies
In the present study the best fit of the agonist-concentration relationships was generally obtained with the sum of two Hill components. The values obtained for the Hill slope with single component fits tended to be lower than with two-component fits. In the case of α4–1β2, fitting the data with a single Hill component gave a shallow Hill slope of <1, immediately suggesting receptor heterogeneity. In many previous studies, EC50 values for nicotinic subunit combinations have been quoted using a
Acknowledgements
We would like to acknowledge the support of The Wellcome Trust, The M.R.C. (UK), The Royal Society, S.H.E.R.T., The Strathclyde Molecular Biology Laboratory, The Strathclyde University Research and Development Fund, and the technical contributions of Fiona Kempsill and Angela Garman. We would also like to thank Professor Steve Heinemann, Drs Jim Boulter and David Johnson (Salk Institute), and Dr Robert Duvoisin (Cornell University) for the provision of AChR subunit cDNAs and Dr Emily Liman,
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