Recovery from Desensitization of Neuronal Nicotinic Acetylcholine Receptors of Rat Chromaffin Cells Is Modulated by Intracellular Calcium through Distinct Second Messengers

Abstract

The mechanisms through which changes in intracellular Ca2+ concentration ([Ca2+]i) might influence desensitization of neuronal nicotinic receptors (nAChRs) of rat chromaffin cells were investigated by simultaneous patch-clamp recording of membrane currents and confocal microscopy imaging of [Ca2+]i induced by nicotine. Increases in [Ca2+]i that were induced by membrane depolarization or occurred spontaneously did not influence inward currents elicited by focally applied test pulses (10 msec) of nicotine, indicating that raised [Ca2+]i per se did not trigger desensitization of nAChRs. Desensitization of nAChRs, evoked by 2 sec focal application of nicotine, which largely raised [Ca2+]i, was not affected by intracellular application of agents that activate or depress protein kinase C (PKC) or A (PKA) or inhibit phosphatase 1, 2 A and B. Conversely, recovery from desensitization was facilitated by the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the phosphatase 2 B inhibiting complex of cyclosporin A-cyclophilin A, whereas it was impaired by the broad spectrum kinase inhibitor staurosporine. The effects of PMA or staurosporine were prevented by the intracellularly applied Ca2+ chelator BAPTA. The adenylate cyclase activator forskolin accelerated recovery, whereas the selective PKA antagonist Rp-cAMPS had an opposite effect. The action of staurosporine and Rp-cAMPS on recovery from desensitization was additive. It is proposed that when nAChRs are desensitized, they become susceptible to modulation by [Ca2+]i via intracellular second messengers such as serine/threonine kinases and calcineurin. Thus, the phosphorylation state of neuronal nAChRs appears to regulate their rate of recovery from desensitization.

Desensitization of neuronal nicotinic receptors

“…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).…”

“…Indeed, if nicotine-induced intracellular Ca 2ϩ transients in chromaffin cells are limited using fast chelation by BAPTA, recovery from desensitization is uniformly fast, even in the presence of PKC inhibition, which under control conditions would markedly slow recovery ( Fig. 4; Khiroug et al, 1998). Additional data showing that inhibition of the Ca 2ϩ -dependent phosphatase calcineurin and stimulation of PKC activity by phorbol ester both promote recovery from desensitization have lead to the hypothesis that Ca 2ϩ regulates desensitization by altering the balance of kinase/phosphatase function-with an increase in cellular phosphorylation (PKA-or PKC-dependent) promoting its recovery (Hardwick and Parsons, 1996;Khiroug et al, 1998;Paradiso and Brehm, 1998;Fenster et al, 1999a).…”

“…4; Khiroug et al, 1998). Additional data showing that inhibition of the Ca 2ϩ -dependent phosphatase calcineurin and stimulation of PKC activity by phorbol ester both promote recovery from desensitization have lead to the hypothesis that Ca 2ϩ regulates desensitization by altering the balance of kinase/phosphatase function-with an increase in cellular phosphorylation (PKA-or PKC-dependent) promoting its recovery (Hardwick and Parsons, 1996;Khiroug et al, 1998;Paradiso and Brehm, 1998;Fenster et al, 1999a). These studies, while supporting a general role for receptor phosphorylation in the regulation of desensitization, do not provide any direct evidence that subunits are modified.…”

“…2). However, if dephosphorylation is strictly state dependent, occurring only when the receptor is desensitized (see Khiroug et al, 1998), then recovery from desensitization would be incomplete, because the relative balance of R, I, and D states would become altered only after desensitization had occurred. In this manner nAChRs could act as coincidence detectors that would associate receptor desensitization with intracellular signals such as Ca 2ϩ (Khiroug et al, 1998;Covernton and Lester, 2002).…”

“…However, if dephosphorylation is strictly state dependent, occurring only when the receptor is desensitized (see Khiroug et al, 1998), then recovery from desensitization would be incomplete, because the relative balance of R, I, and D states would become altered only after desensitization had occurred. In this manner nAChRs could act as coincidence detectors that would associate receptor desensitization with intracellular signals such as Ca 2ϩ (Khiroug et al, 1998;Covernton and Lester, 2002). Katz and Thesleff (1957, p 68) in their original observations noted that "especially after prolonged large doses, recovery was slowed or incomplete," and Boyd (1987) extended these observations by remarking on both a temperature-dependence, for which a possible phosphorylation step was invoked, and a complete loss of function after several days in agonist.…”