Elsevier

Brain Research

Volume 882, Issues 1–2, 3 November 2000, Pages 155-168
Brain Research

Research report
Choline and acetylcholine have similar kinetic properties of activation and desensitization on the α7 nicotinic receptors in rat hippocampal neurons

https://doi.org/10.1016/S0006-8993(00)02863-8Get rights and content

Abstract

The α7-type nicotinic acetylcholine receptor (nAChR) was recently found to be both fully activated and desensitized by choline, in addition to ACh. In order to understand the combined effects of the two agonists on α7 nAChR-mediated neuronal signaling, the kinetics of the receptor-channel’s interaction with ACh and choline was examined. To this end, whole-cell and single-channel currents evoked by fast-switching pulses of the agonists were recorded in rat hippocampal neurons in culture. Currents evoked by equieffective concentrations of choline and ACh were very similar, except that choline-evoked currents decayed more quickly to the baseline after removal of the agonist, and that recovery from desensitization was faster with choline. The conductance of channels activated by choline and ACh was 91.5±8.5 and 82.9±11.6 pS, respectively. The mean apparent channel open times were close to 100 μs, with both agonists. After a 4-s exposure to concentrations up to 80 μM ACh or 600 μM choline, the extent of desensitization and the cumulative charge flow carried by the channels increased in the same proportion, until reaching a maximum. At higher concentrations of either agonist, the cumulative charge started decreasing with concentration, reflecting further desensitization. Kinetic modeling suggested that α7 nAChRs have at least two non-equivalent paths to desensitized states, and that choline dissociates faster than ACh from the receptor. Our results established that the main difference between choline and ACh is of affinity, and support the concept that the switching of endogenous agonist may change the desensitization–resensitization dynamics of α7 nAChRs.

Introduction

A large body of evidence has been accumulated indicating that there is a direct participation of nicotinic acetylcholine receptors (nAChRs) in the control of neuronal function in the central nervous system, particularly in the hippocampus [2], [3], [4], [6], [10], [18], [19], [23], [37]. These receptors are located both postsynaptically in fresh hippocampal tissue [4], [18], [22] and presynaptically, where they act by modulating transmitter release [1], [5], [7], [19], [20], [23]. While more than one nAChR subtype is expressed in the hippocampus, sometimes with co-expression in single neurons [3], [12], [37], the α7-type nAChRs are by far the most abundant in cultured neurons [3], [4], [18]

An important advance in understanding the function of nAChRs in the CNS was the demonstration that choline, a precursor and metabolite of ACh, is an effective agonist of α7 nAChRs [6], [26], [29]. It was recently demonstrated that human cortical interneurons express α7 nAChRs that are also effectively activated by choline [8]. Similar to ACh, choline is an efficacious desensitizer, with significant effects observed at concentrations above 10 μM [6]. This is close to the mean extracellular concentration of choline in the brain, which is fairly stable around 4 to 6 μM [25]. Because of the dual activating/inhibiting effect, the physiological roles of ACh and choline must depend on the detailed dynamics of their concentrations (the amount of released transmitter, the velocity of diffusion, the rate of hydrolysis, and the rate of choline uptake), and also on the spatial distribution and subcellular localization of the receptors, and their kinetics. There is novel data suggesting that native α7 nAChRs differ structurally from other mammalian ionotropic receptors in being homopentamers [14], [16]. This opens the possibility of there being up to five agonist binding sites and adds considerable complexity to the kinetic behavior of the receptor-channel.

After the activation of cholinergic neurons, the concentration of choline is expected to rise sharply in the areas near ACh release sites due to the rapid hydrolysis of ACh. This elevation of choline concentration surely affects the responsiveness of the nicotinic receptors to ACh. It could isolate the nicotinic response in a spatial and temporal manner, by causing desensitization of receptors in the neighborhood of the ACh release site. It is also possible that choline is the only agonist to reach α7 nAChRs located away from the release sites, providing a more prolonged (and less intense) signal that would reflect the cumulative cholinergic activity of the area. It appears that the neuronal α7 nAChRs have two effective endogenous agonists that may act simultaneously or in tandem, competing for the same binding sites. Thus, to understand the physiological significance of this condition, we investigated the kinetics of the currents evoked by both endogenous agonists.

Analyses of whole-cell currents mediated by α7 nAChRs have indicated that the kinetics of receptor activation and inactivation are strongly dependent on agonist concentration [3]. Single-channel studies of both native receptors [12] and ectopically expressed mammalian α7 nAChRs [9] revealed relatively very short channel open times, fast rate of desensitization, and large single-channel conductance when ACh was the agonist. However, to the best of our knowledge, the properties of choline-evoked single-channel currents in neurons have not been reported before. Here, we explored the kinetic properties of α7 nAChR-gated currents recorded in whole-cell and outside-out configurations of the patch-clamp technique, comparing the currents evoked by the two endogenous agonists, ACh and choline. The quantitative observations were then used to build kinetic models that would aid in the interpretation of the available data on α7 nAChRs located in the brain and that would also generate specific hypotheses to be tested in future experiments.

Section snippets

Cell culture

Hippocampal neurons from Sprague-Dawley rats were cultured as described previously [3]. Briefly, hippocampi of 17–18-day-old fetuses were dissected; the cells were mechanically dissociated after trypsinization (30 min, 0.25% trypsin, Gibco) and plated on collagen-coated culture dishes. The cells were maintained in Minimum Essential Medium (MEM, Gibco) containing 10% heat-inactivated horse serum and 2 mM glutamine. One week after plating, glial cell proliferation was inhibited with

Results

In the first part of this section we will present the comparative characterization of currents evoked by the two endogenous agonists of α7 nAChRs, ACh and choline. In the second part we will attempt to explain these results in terms of kinetic models: experiments described there were designed to answer specific questions about possible kinetic mechanisms underlying α7 nAChR-mediated currents.

Single-channel currents mediated by the two natural agonists of α7 nAChRs

The present investigation describes for the first time the single-channel currents activated by choline in mammalian neurons. These currents had very similar properties to those activated by ACh and, based on several arguments, both can be attributed to the native hippocampal α7 nAChRs, as previously proposed [12]. Whole-cell currents activated by choline in hippocampal neurons were completely blocked by α-bungarotoxin and methyllycaconitine, both in isolated cultured cells [6] and in fresh

Acknowledgements

This study was supported by USPHS grants NS25296 and ES05730, and by PRONEX/MCT and CNPq (from Brazil). A.M. was a Fogarty Fellow (TW05389). The technical assistance of Mr. Benjamin Cumming, Mrs. Barbara Marrow, and Ms. Mabel Zelle is gratefully acknowledged. The authors wish to thank Dr. Yasco Aracava for valuable discussions regarding the manuscript.

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    1

    Present address: Department of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67, H-1450 Budapest, Hungary.

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