Acetylcholine receptor extracellular domain determines sensitivity to nicotine-induced inactivation

Abstract

We have shown previously that chronic exposure to submicromolar concentrations of nicotine permanently inactivates α4β2 and α7 neuronal nicotinic acetylcholine receptors while α3β2 acetylcholine receptors are resistant to inactivation. Phosphorylation of the large cytoplasmic domain has been proposed to mediate functional inactivation. Chimeric subunits consisting of human α4 sequence from their N-terminus to either the beginning of the first transmembrane domain or the large cytoplasmic domain and α3 sequences thereafter formed acetylcholine receptors with β2 subunits which were as susceptible to nicotine-induced inactivation as wild-type α4 acetylcholine receptors. The converse chimeras, containing the N-terminal parts of the α3 subunit and the C-terminal parts of the α4 subunit, formed acetylcholine receptors with β2 subunits which were as resistant to nicotine-induced inactivation as wild-type α3β2 acetylcholine receptors. Thus, inactivation of acetylcholine receptors produced by chronic exposure to nicotine results primarily from effects of the agonist on the extracellular and transmembrane domains of the α subunit.

Introduction

Nicotine, acting at neuronal nicotinic acetylcholine receptors, is the primary component of tobacco that drives its habitual use (Benowitz et al., 1990). Nicotinic agonists being developed as drugs to treat Alzheimer's disease, Parkinson's disease or chronic pain Bannon et al., 1998, Holladay et al., 1997 may have some of the same effects on acetylcholine receptors as does chronic exposure to nicotine. Along with the synchronized activation of acetylcholine receptors by a rapid bolus of nicotine that results from smoking a cigarette, chronic nicotine exposure differentially affects both the amount and function of neuronal acetylcholine receptor subtypes. Acetylcholine receptors containing β2 subunits are especially important in developing dependence on nicotine, as shown by experiments with β2 knockout mice (Picciotto et al., 1998). The number of high affinity nicotine binding sites, especially α4β2 acetylcholine receptors, is increased up to fourfold in the brains of tobacco smokers and animals chronically treated with nicotine Wonnacott, 1990, Collins and Marks, 1996, Flores et al., 1997, Perry et al., 1999. In addition, chicken α4β2 acetylcholine receptors expressed in Xenopus oocytes or in a permanently transfected cell line doubled in amount when chronically exposed to nicotine (Peng et al., 1994). The EC₅₀ for upregulation was 0.2 μM, essentially equal to the concentration of nicotine typically found in the serum of smokers (Benowitz et al., 1990). Chronic exposure to 10 μM nicotine not only reversibly desensitized these acetylcholine receptors, but also permanently inactivated many of them (Peng et al., 1994). Similarly, human α4β2 acetylcholine receptors in a permanently transfected cell line were upregulated 15-fold by chronic nicotine exposure, but the ion flux activity increased only 45%, thus the amount of acetylcholine-induced ion flux per acetylcholine receptor was decreased (Gopalakrishnan et al., 1996). α3-Containing acetylcholine receptors are also upregulated, although much higher concentrations of nicotine are required. A mixture of α3-containing acetylcholine receptors expressed by the human neuroblastoma cell line SH-SY5Y increased by 600% in response to exposure to high concentrations of nicotine (Peng et al., 1997), and functional human α3β2 and α3β2α5 acetylcholine receptors in permanently transfected cell lines were increased up to 24-fold by chronic exposure to high concentrations of nicotine (Wang et al., 1998).

Chronic nicotine exposure causes inactivation of some acetylcholine receptor subtypes Collins and Marks, 1996, Dani and Heinemann, 1996, Gopalakrishnan et al., 1996, Hsu et al., 1996, Fenster et al., 1997, Fenster et al., 1999, Olale et al., 1997, Ke et al., 1998. Using X. laevis oocytes as an expression system, we (Olale et al., 1997) have previously shown that 0.2 μM nicotine irreversibly inactivates most human α4β2 and α7 acetylcholine receptors but inhibits α3β2 acetylcholine receptors much less and more reversibly. This means that although α3 acetylcholine receptors are present in lower amounts in the brain than are α4 and α7 acetylcholine receptors, after chronic exposure to nicotine, α3 acetylcholine receptors may be able to play a greater role in acute responses to endogenous acetylcholine or subsequent doses of nicotine. α3 acetylcholine receptors also play a major role in synaptic transmission in peripheral autonomic ganglia. Tolerance for nicotine exhibited by tobacco users, as well as the behavioral effects of nicotine, may well reflect the sustained inhibition of α4 and α7 acetylcholine receptors in combination with the residual susceptibility of α3-containing acetylcholine receptors and related acetylcholine receptors. This hypothesis does not depend solely on studies of expressed cloned acetylcholine receptor subtypes. Rat striatal synaptosomes also show long-lasting inactivation of nicotinic acetylcholine receptor function after chronic treatment with micromolar concentrations of nicotine Collins and Marks, 1996, Rowell and Duggan, 1998. Rowell and Duggan (1998), for instance, reported reversible desensitization after 10 min exposures to 0.3 μM nicotine but complete recovery within 1 h. However, 12 s of 30 μM nicotine resulted in 30% of inactivation not reversed after 1 h. They concluded that 50% of nicotine-induced dopamine release was due to α3β2 acetylcholine receptors that were not inhibited significantly, while the other half was due to α4β2 acetylcholine receptors that were mostly blocked.

It is not clear what molecular mechanisms are involved in the functional inactivation or upregulation associated with chronic exposure of acetylcholine receptors to nicotine. It is also unclear whether the two phenomena are the result of a single process. Conceivably, chronic exposure of acetylcholine receptors to nicotine may lead to a conformational change, altering ligand binding sites on the extracellular surface or affecting the transmembrane linkage to the channel gate near the cytoplasmic surface or other parts of the cytoplasmic surface. The effects of chronic nicotine exposure may instead or in addition be a result of covalent modification of the large cytoplasmic domain by mechanisms like phosphorylation, as has been proposed by Hsu et al. (1997) and Eilers et al. (1997). Acetylcholine receptor subunits contain potential phosphorylation sites in their large cytoplasmic domains (Miles and Huganir, 1988). Indeed, sequence analysis of α4 acetylcholine receptors subunits shows consensus sequences for phosphorylation by protein kinase A and tyrosine-specific protein kinase within the large cytoplasmic domain, and a protein kinase C phosphorylation site has been reported to regulate transitions between “shallow” and “deep” desensitized states (Fenster et al., 1999). α3 Acetylcholine receptor subunits have similar, but not identical, sites (Heinemann et al., 1991).

In this paper we investigate whether the extracellular domain, the transmembrane domains M1–M3 or the large cytoplasmic domain of α4 subunits play a direct role in the nicotine-induced functional inactivation of human α4β2 acetylcholine receptors. Nicotinic acetylcholine receptors are formed from five homologous subunits organized around a central cation channel Karlin and Akabas, 1995, Wilson and Karlin, 1998, Lindstrom, in press, Lindstrom, 1996. Those formed from α7, α8 or α9 subunits can function as homomers. Acetylcholine receptors containing α2, α3, α4 or α6 subunits require β2 or β4 subunits and may also contain α5 or β3 subunits. Muscle acetylcholine receptors are thought to have their subunits organized in the order α1γα1δβ1 or α1εα1δβ1. Neuronal acetylcholine receptors are thought to similarly alternate α and β subunits so as to form two acetylcholine receptor binding sites at the interfaces between one side of each of two α subunits with the adjacent β subunit, e.g., α4β2α4β2β2 Karlin, 1993, Lindstrom, in press. Acetylcholine receptor subunits exhibit signal sequences at the N-terminus that are cleaved during translation and that serve to target the large N-terminal domain to the extracellular surface. This extracellular domain precedes the putative transmembrane domains M1, M2 and M3. M1 is thought to provide part of the channel lining and may act as a linkage between agonist binding in the extracellular domain and the channel gate formed by the loop between M1 and M2. M2 provides most of the cation channel lining. In between M3 and M4 is the large cytoplasmic domain containing possible phosphorylation sites that may be involved in regulating acetylcholine receptor function, turnover or location. It is the most variable region of sequence among types of acetylcholine receptor subunits and among species for a given type of subunit. M4 comes immediately after the large cytoplasmic domain, and a small C-terminal extracellular domain is located at the end of each subunit.

α3 and α4 acetylcholine receptors exhibit different susceptibilities to nicotine-induced functional inactivation, and it is conceivable that these differences could be due to differences in their large cytoplasmic domains, such as differences in phosphorylation Eilers et al., 1997, Hsu et al., 1997, Fenster et al., 1999. While most α4β2 acetylcholine receptors are irreversibly inactivated by a 24-h treatment with 0.2 μM nicotine, most α3 type acetylcholine receptors remain functional Hsu et al., 1996, Olale et al., 1997, Wang et al., 1998. By constructing chimeras in which domains of the α3 and α4 subunits have been switched, and then testing these chimeras for nicotine-induced functional inactivation, we can determine which domains of the α subunit determine susceptibility to the effects associated with chronic nicotine exposure. If only the large cytoplasmic domain determined this susceptibility, this would be consistent with a dominant role for phosphorylation of this part of the α subunit. However, if the large cytoplasmic domain were not sufficient to determine susceptibility to the effects of chronic nicotine exposure, then phosphorylation of the large cytoplasmic domain would not be the primary determinant and the relevant region would lie N-terminal to the cytoplasmic domain. In this case, conformational changes of the acetylcholine receptor associated with the extracellular domain or channel could provide plausible explanations of the mechanisms involved, as might covalent modification of the gate region between M1 and M2.