Desensitization of nicotine acetylcholine receptors: Modulation by kinase activation and phosphatase inhibition

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Abstract

The desensitization of α-bungarotoxin-insensitive native neuronal nicotinic receptors was studied in rat cortical cell cultures using the patch clamp technique. Thirty-minute perfusions of nicotine reduced currents evoked by short test pulses of 300 μM acetylcholine over a range of 3 to 300 nM, with an IC50 of 51 nM. The time course of desensitization onset was fit by a biexponential function consisting of a fast time constant of about 1 min and a slower component of 6–10 min. The desensitization recovery process was also biexponential and was dominated by a slow time constant of 12–20 min, as well as a minor component of about 1 min. The intracellular dialysis of either the protein kinase C activator phorbol-12-myristate-13 acetate or the phosphatase inhibitor cyclosporin A accelerated the desensitization recovery rate by 2-fold. The data imply that endogenous cortical nicotinic receptor channels may enter one of two desensitization states. The first state (D1) is characterized by rapid entry and recovery, whereas transitions into and out of the second state (D2) occur at slower rates. The D2 receptor state may arise by a sequential transition from the D1 conformation. Protein kinase C activation or phosphatase 2B inhibition may favor the D1 receptor state over that of D2 to promote faster overall rates of desensitization recovery.

Introduction

Since the “discovery” in pre-Columbian America nearly 500 years ago, tobacco-related products have spread to nearly every corner of the globe and are regularly used by 1.1 billion smokers. However, it is only within the last 30 years that science has begun to understand the molecular mechanisms of how nicotine, the primary constituent of tobacco, exerts its effects on the brain. It is now clear that nicotine must be interacting with one or more of the multiple subtypes of neuronal nicotinic acetylcholine receptors that are formed from various homo- and heteromeric combinations of α and β nicotinic subunits (McGehee and Role, 1995). Perhaps the most pertinent neuronal nicotinic receptors related to nicotine use are those composed of the α4β2 subunit combination. The α4β2 nicotinic receptor is the predominant high-affinity nicotine binding receptor in the mammalian brain (Flores et al., 1992). Furthermore, α4β2 nicotinic receptor densities increase following chronic exposure to low concentrations of nicotine—both in vivo (Flores et al., 1992) and in vitro (Peng et al., 1994, Whitaker et al., 1998). Such characteristics mark them as prime mediators of the effects of tobacco on the central nervous system.

Pharmacokinetic studies of smokers indicate that venous nicotine concentrations gradually increase over the course of a day as a series of “peaks and troughs” until a steady-state plateau is reached (Russell, 1987). However, even in the heaviest smokers these plateaus rarely reach 300 nM. Even the measured “per puff” arterial nicotine bolus reaching the brain from the lungs is usually less than 1 μM. (Henningfield et al., 1993). In cultured cortical cells, currents evoked by the acute application of 300 nM nicotine are less than 5% of the maximally attainable acetylcholine-induced current (Marszalec et al., 1999). However, a 10 min perfusion of 300 nM nicotine desensitizes acetylcholine test responses by more than 60%. Such in vivo and in vitro observations have prompted speculation that some behavioral effects related to tobacco use arise from α4β2 nicotinic receptor desensitization (Balfour, 1994).

In this study, we have recorded and analyzed the nicotine-induced desensitization of α-bungarotoxin-insensitive acetylcholine-evoked currents in rat cortical neurons. In addition to studying the magnitude and temporal aspects of nicotine-induced desensitization, additional experiments were conducted to determine whether agents modifying the activities of intracellular protein kinase C or phosphatase 2B might alter the character of this desensitization. This is in keeping with literature reports that agents that modify protein phosphorylation also modify nicotinic receptor desensitization (Swope et al., 1999). Studies in cell lines and transfected Xenopus oocytes show that α4β2 receptor desensitization is affected by either protein kinase C activation or phosphatase inhibition (Eilers et al., 1997, Khiroug et al., 1998, Fenster et al., 1999). We wanted to determine whether this was also true for α4β2-like nicotinic receptors in native cortical neurons. The use of cultured neurons allows experiments of nicotinic receptors in their native membrane environment, while avoiding some of the problems arising from the transfection of these receptors in foreign hosts (Lewis et al., 1997, Sivilotti et al., 1997, Buisson et al., 2000).

Section snippets

Primary cultures of cortical neurons

Cortical neurons were prepared from 17-day embryonic Sprague–Dawley rat pups removed under methoxyflurane anesthesia as approved by the Northwestern University Animal Care and Use Committee. The brains were dissected in calcium- and magnesium-free phosphate buffered saline solution (PBS). Small wedges of frontal cortex were placed in PBS containing 0.25% (w/v) trypsin (type XI, Sigma-Aldrich, St. Louis, MO) and incubated for 20 min at 35 °C. Afterwards, the tissue was triturated by repeated

Acetylcholine-induced currents in cultured cortical neurons

Data previously reported by this laboratory (Aistrup et al., 1999) have shown that two types of acetylcholine-induced currents can be recorded from cortical neurons. One type of acetylcholine-evoked response rapidly desensitized (τ  20 ms) at high concentrations of agonist and was inhibited by nanomolar levels of α-bungarotoxin. The second type of current desensitized more slowly (τ  5 s) at high levels of agonist, was unaffected by α-bungarotoxin, but was antagonized by dihydro-β-erthroidine.

Discussion

This study extends previous descriptions of nicotinic receptor desensitization (Fenster et al., 1997, Fenster et al., 1999) to include those α-bungarotoxin-insensitive α4β2-like receptors endogenous to rat cortical neurons. The desensitization here was evoked by nicotine concentrations similar to those encountered with tobacco use. The original model of motor end-plate activation by Katz and Thesleff (1957) postulated that agonist (A) binding to nicotinic receptor channels (R) either induced or

Acknowledgements

We thank Nayla Hasan for her preparation and maintenance of the cortical cell cultures used in this study and Julia Irizarry for secretarial assistance. This work was supported by a National Institutes of Health Grant AA07836.

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