Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract

To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µM nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration‐dependent manner with IC50 values in the 10 nM range following incubation for 24 h and in the 1 nM range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 nM nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first‐order process for both subtypes; half‐lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first‐order for both subtypes; half‐lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor‐mediated neurotransmitter release.

Desensitization of neuronal nicotinic receptors

“…Moreover, functional studies designed to replicate the radiolabeled ligand binding assays find that ␣4 subunit-containing nAChRs are desensitized by much lower concentrations of agonist than those containing ␣3 subunits. The IC 50 values for desensi-tization of functional channels by nicotine/ACh are in the same range as their binding K d 's, Ϸ0.1-10 nM and Ϸ150 -400 nM for ␣4* and ␣3* nAChRs, respectively (Hsu et al, 1996;Fenster et al, 1997), thus confirming the difference in affinity of the "D" state for these receptors. In addition, chimeric ␣3/␣4 subunits (containing the N-terminal agonist binding domain of ␣3 subunits), when expressed with ␤2 subunits, create receptors with an ␣3-like desensitization affinity (IC 50 Ϸ 700 nM) compared to ␣4␤2 (IC 50 Ϸ 50 nM), implying that the ␣ subunit is the primary determinant of affinity (Kuryatov et al, 2000).…”

“…2). Likewise, it is probable that desensitization of the lower affinity putative ␣3␤4*-nAChRs, as observed in various cells representative of both the autonomic nervous system and CNS (Higgins and Berg, 1988;Oortigiesen and Vijverberg, 1989;Mathie et al, 1990;Khiroug et al, 1997Khiroug et al, , 1998Boyd, 1987;Ifune and Steinbach, 1993;Lester and Dani, 1995), in addition to systems designed to specifically express ␣3␤4 receptors (Cachelin and Jaggi, 1991;Hsu et al, 1996;Fenster et al, 1997;Wang et al, 1998;Xiao et al, 1998), can also be explained in terms of the two-state model, although due to the slower time course of desensitization for ␤4 subunit-containing receptors (see below), the biphasic nature may only be seen clearly during longer agonist applications (e.g., Lester and Dani, 1995). Concentration-dependent analyses of desensitization of presumed native ␣3␤4* nAChRs have estimated the affinity of the desensitized state for nicotine to be Ϸ20 -300 nM (Higgins and Berg, 1988;Lester and Dani, 1995).…”

“…Occupancy of these deep desensitized states by nicotine could serve as the trigger for up-regulation in receptor number (Dani and Heinemann, 1996;Fenster et al, 1999b), shown to occur in vivo for ␣4␤2 nAChRs during chronic nicotine exposure (Marks et al, 1983;Schwartz and Kellar, 1985;Flores et al, 1992;Balfour, 1994); this again could potentially involve biochemical steps, because chronic nicotine promotes enhanced ␣4 subunit phosphorylation (Hsu et al, 1997). While controversy exists over whether prolonged agonist exposure results in up-or down-regulation of receptor function (Buisson and Bertrand, 2002), recovery from this "state," whether it requires purely time (slow transition rates) or de novo receptor synthesis (Boyd, 1987;Hsu et al, 1996), likely influences synaptic transmission for prolonged periods. Girod and Role (2001) recently observed that presynaptic nAChR function could be lost for Ͼ24 h following 24 -72 h treatment with low levels of nicotine (Fig.…”

Nicotinic acetylcholine receptors as targets for antidepressants

“…Further complicating matters, degrees of nAChR activation, desensitization and persistent inactivation are subtype-, time-, and dose-dependent, reflecting both form and route of administration (bolus vs continuous), influences that make it difficult to extricate roles of nAChR in mood control. 64,72 Long exposures to low concentrations of agonist will favor deeper levels of desensitization, and this situation is often the case for smokers, who maintain low concentrations of nicotine throughout the day. 71,[73][74][75] With this in mind, it has been proposed that nicotine's predominant effect on many nAChR subtypes over time (its time-averaged effect) is that of an antagonist.…”

“…76 However, some nAChR subtypes are more resistant to desensitization than others, and there is some evidence that certain receptor subtypes become more sensitive to repeated agonist exposure. 72 The most abundant forms of nAChR in the mammalian brain contain either ␣4 and ␤2 subunits or ␣7 subunits. 77,78 However, less abundant nAChR subtypes may play disproportionate roles in some brain functions, and there are some brain regions that have non-␣7 and -␣4␤2 agonist and antagonist profiles.…”