Nicotine trapping causes the persistent desensitization of α4β2 nicotinic receptors expressed in oocytes

Abstract

To determine whether prolonged nicotine exposure persistently inactivates rat a4b2 nicotinic receptors expressed in Xenopus oocytes, we measured the voltage-clamped a4b2 response to acetylcholine (ACh) before and 24 h after, 1-h or 12-h incubations in 10 lM nicotine. A 12-h incubation in 10 lM nicotine depressed the a4b2 ACh response for 24 h without affecting total or surface a4b2 expression. To determine whether oocyte-mediated nicotine release caused this depression, we co-incubated an a4b2-expressing oocyte with an un-injected one (pre-incubated in 10 lM nicotine for 12 h) for 24 h and measured the change in the a4b2 ACh response. The response decreased by the same factor after the co-incubation as it did after a 12-h incubation in 10 lM nicotine and a 24-h incubation in nicotine-free media. Thus, oocytemediated nicotine release caused the persistent desensitization we observed after a 12-h incubation in 10 lM nicotine. Prolonged nicotine exposure persistently depresses the agonist response of a4b2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes (Peng et al. 1994;Hsu et al. 1996;Olale et al. 1997;Kuryatov et al. 2000). For example, incubations in 0.05-10 lM nicotine for 1-6 days reduce the response of a4b2 nAChRs expressed in Xenopus oocytes by 40-80% for a period of 24-48 h after the incubation ends (Peng et al. 1994;Hsu et al. 1996;Olale et al. 1997;Kuryatov et al. 2000). The molecular mechanism that underlies this persistent depression has not been established. However, it is thought to involve some kind of post-translational modification of the receptor (reviewed in Ochoa et al. 1989). It does not require persistent nicotine binding because [ 3 H]nicotine dissociates from rat brain nAChRs with a time constant of 2.5 min at 22°C (Lippiello et al. 1987).In contrast to oocytes, incubation in 0.1-10 lM nicotine for 1-7 days does not persistently inhibit the agonist response of a4b2 nAChRs expressed in mammalian cell lines (Gopalakrishnan et al. 1996;Buisson and Bertrand 2001). For example, a 7-day incubation in 100 nM nicotine increases the maximum ACh-induced 86 Rb influx from HEK cells expressing a4b2 nAChRs by 45% (Gopalakrishnan et al. 1996), and a 24-h incubation in 10 lM nicotine increases the maximum voltage-clamped ACh response of HEK cells expressing a4b2 nAChRs by 63% (Buisson and Bertrand 2001). Consistent with these increases, twice daily injections of nicotine tartrate (2 mg/kg) for 10 days 3 H]nicotine concentration at time t; NR, peak post-incubation a4b2 ACh response normalized to the pre-incubation value; NR 1 , final steady-state normalized response for recovery from ACh-induced desensitization; P solute , solute permeability coefficient; P nicotine , P solute for nicotine; t, time; s D , time constant for diffusion between two compartments; V cell , intracellular volume.

Rare Human Nicotinic Acetylcholine Receptor α4 Subunit (CHRNA4) Variants Affect Expression and Function of High-Affinity Nicotinic Acetylcholine Receptors

“…This effect opposes the actions of those variants on receptor assembly in the absence of nicotine. A confounding factor comes from the observation that X. laevis oocytes act as "nicotine sponges" during incubation with nicotine and release desensitizing concentrations of nicotine into superfusion buffer during recording (Jia et al, 2003). However, HS and LS forms of a4b2 nAChRs do not differ in their sensitivity to steady-state desensitization with subactivating concentrations of nicotine (Marks et al, 2010), so any differences in function observed after 24-hour nicotine across the a4 variants are due to effects conferred by the variants themselves.…”

Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human α4β2 Acetylcholine Receptors

“…Nicotine crosses cell membranes very quickly (Fig. 9), and in some cells, it can accumulate to much higher than ambient concentrations (e.g., in negatively charged compartments within X. laevis oocytes) (Jia et al, 2003;A. Kuryatov, V. Gerzanich, and J. Lindstrom, unpublished data).…”

“…This probably reflects irreversible desensitization of some of the AChRs (Gentry and Lukas, 2002;Gentry et al, 2003). An alternate explanation would be that nicotine, which had accumulated within the cells, partitioned out slowly, sustaining the AChRs in a reversibly desensitized state (Jia et al, 2003).…”

α4β2 Nicotinic Receptors with High and Low Acetylcholine Sensitivity: Pharmacology, Stoichiometry, and Sensitivity to Long-Term Exposure to Nicotine

“…The reporter mutation approach was not used to monitor changes in the function of ␣4␤2 nAChRs because the leftward shifts in the ACh concentration-response curves caused by the reporter mutation or by changes in subunit composition would overlap, making it difficult to separate both effects (compare curves in Fig. 2 (Jia et al, 2003;López-Herná ndez et al, 2004) the ACh responses of oocytes expressing ␣4␤2 nAChRs were depressed by long-term exposure to 5 M nicotine. Therefore, before recordings, oocytes were maintained for 4 h in 15 ml of nicotine-free Barth's solution in a Petri dish (one oocyte per Petri dish) placed on a rotating platform.…”