Acetylcholine receptor desensitization induced by nicotine in rat medial habenula neurons

Abstract

1. The activation and desensitization properties of nicotinic acetylcholine receptor (nAChR) channels were examined in acutely isolated medial habenula (MHb) neurons using whole cell patch-clamp recordings. nAChR-mediated currents were evoked by applying known concentrations of nicotinic agonists using rapid solution exchange techniques. 2. At a membrane potential of -60 mV, nAChR currents were observed above a concentration of approximately 100 mM nicotine. The peak current amplitude at low doses of agonist was proportional to the square of the concentration of nicotine, indicating that at least two molecules of agonist were required for channel opening. The concentration of nicotine required for half-maximal nAChR activation was estimated as 77 microM from a complete concentration-response curve. 3. During the continuous activation (2-5 s) of nAChRs by high concentrations of nicotine (300 microM), the current desensitized rapidly and extensively. The desensitization phase was described by the sum of two exponentials, with time constants of 210 and 1,435 ms. The fast component comprised 74% of the desensitizing phase of the current. Recovery from desensitization induced by 2- s applications of 300 microM nicotine was also fast and could be reasonably well described by a single exponential with a time constant of approximately 800 ms. Both the time courses of desensitization and recovery from desensitization were slightly slower at positive membrane potentials. 4. Incubation of neurons with low concentrations of nicotine (100 nM-10 microM) caused a slowly developing but pronounced desensitization of the nAChRs. In these cases desensitization was assessed from the reduction in the amplitude of the peak nicotinic current induced by repetitively applied pulses of a higher test concentration of agonist. A 5-min continuous exposure to 1 microM nicotine reduced the amplitude of the acetylcholine (30 microM, 1 s) test response to < 30% of its control value. As with higher concentrations of nicotine, the onset of the desensitization induced by 1 microM nicotine was biexponential, with fast and slow time constants of 15 s and 1.74 min, respectively. Recovery from the desensitization induced by these longer applications of nicotine was much slower than that observed with the brief pulses of high concentrations of nicotine. The concentration required for half-maximal desensitization after a 5-min incubation was approximately 300 nM. 5. Peak nAChR currents were approximately 85% smaller at +40 mV compared with -40 mV. The receptors that do not open at positive potentials desensitize almost as well as they would at negative potentials after channel opening.(ABSTRACT TRUNCATED AT 400 WORDS)

Desensitization of neuronal nicotinic receptors

“…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). The slightly higher apparent affinities compared to those from the CNS binding studies (e.g., Whiteaker et al, 2000) could reflect the assembly of more complex heteromeric receptors in autonomic ganglia (Conroy and Berg, 1995;see below).…”

“…However, in Xenopus oocytes accumulation and slow-release of permeable agonists, for example, nicotine (Fenster et al, 1999b), may dramatically distort recovery kinetics, possibly due to rebinding and prolonged stabilization of desensitized states of high affinity receptors, for example, ␣4␤2 nAChRs (Jia, Flotildes, Li, and Cohen, unpublished observations). When examined in small cell systems, including neurons, recovery is fairly complete following short periods of agonist exposure for both ␣3 and ␣4 subunit expressing receptors (e.g., Boyd, 1987;Row-ell and Hillebrand, 1994;Lester and Dani, 1995;Booker et al, 1998; see Table 1). …”

“…However, the desensitization kinetics of native channels often tend to be faster than these simple ␣␤ pairs. For example, the major component of desensitization of presumed ␣3␤4* receptors in rat medial habenula cells (Quick et al, 1999) by 300 M nicotine occurs with an exponential time constant, t Ϸ 200 ms (Lester and Dani, 1995) compared with t Ϸ 1-2 s for ␣3␤4* receptors expressed in HEK cells . This apparent discrepancy may be partially explained by the inclusion of an additional ␣5 subunit; ␣5␣3␤4 receptors desensitize approximately threefold faster (Gerzanich et al, 1998;Quick et al, 1999) than ␣3␤4 nAChRs, and ␣5 is often found associated with ␣3␤4 receptors (Conroy and Berg, 1995;Sheffield et al, 2000).…”

Invertebrate models of drug abuse

“…Upon binding of acetylcholine, nAChRs undergo conformational changes to transiently open a cation-selective channel, resulting in depolarization of the neuron. Subsequently, the ion channel closes, and the receptor is temporarily refractory to agonist (Lester and Dani, 1995). Long-term nicotine exposure can cause upregulation of nAChRs (Pauly et al, 1996).…”

Inaugural Article: Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method

“…Receptor isomerizes to a transient, fast-onset desensitized state on the 0.1-to 10-s time scale and to a stable, slow-onset desensitized state on the 10-to 100-s time scale (10)(11)(12)(13)(14)(15)(16)(17). The ACh affinities of the transient, fast-onset and stable, slow-onset desensitized states are, respectively, 2 and 4 orders of magnitude greater than the affinity of the resting state (18,19).…”