Multiple components in the agonist concentration–response relationships of neuronal nicotinic acetylcholine receptors

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Abstract

Assessing the potential of nicotinic agonists as therapeutic agents has frequently relied upon single component EC50 values obtained from studies of nicotinic receptors expressed in Xenopus oocytes. We have evaluated the validity of this approach using voltage clamp techniques. In general, agonist concentration–response plots for the α3β2, α3β4, α4–1β2, α4–1β4 and α7 combinations were poorly fitted by a single component Hill-equation. Improved fits were obtained with the sum of two components, although only in the case of α3β4 and α4–1β2 was the improvement significant regardless of the weighting method used. For the acetylcholine (ACh) concentration–response relationships of the α4–1β2 combination, the two EC50 values were 0.3 and 58.3 μM. For the α3β4 combination, the two EC50 components were 39 and 2919 μM. The 39 μM component of α3β4 represented 36% of the sum of the maximum responses of both curves. This shows that for some combinations, the secondary components represent a well-separated, major population of receptors. Therefore, published EC50 values which assume that only a single subtype of functional receptor is present may not accurately describe agonist action may therefore need to be re-evaluated.

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,

References (35)

  • E. Cooper et al.

    Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor

    Nature

    (1991)
  • P.J.O. Covernton et al.

    Differential modulation of neuronal nicotinic receptor subtypes by ethanol

    Br. J. Pharmacol.

    (1997)
  • P.J.O. Covernton et al.

    Comparison of neuronal nicotinic receptors in rat sympathetic neurones with subunit pairs expressed in Xenopus oocytes

    J. Physiol.

    (1994)
  • S. Fucile et al.

    α5 subunit forms functional α3β4α5 nicotinic acetylcholine receptors in transfected human cells

    Neuroreport

    (1997)
  • A.J. Gibb et al.

    Expression of cloned receptor subunits produces multiple receptors

    Proc. R. Soc. Lond.

    (1990)
  • S.A. Glantz

    Primer of Biostatistics

    (1992)
  • M. Gopalakrishnan et al.

    Nicotine: therapeutic prospects?

    Pharma. News

    (1998)
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