Cyclic AMP-dependent phosphorylation of a neuronal acetylcholine receptor alpha-type subunit

Abstract

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate synaptic transmission through the ganglion. A cAMP- dependent process has previously been shown to enhance the ACh response of the neurons 2- to 3-fold without requiring the synthesis of new receptors. We show here that the receptors can be phosphorylated in situ by a cAMP-dependent process. The phosphorylation occurs predominantly on components of 50 and 58 kDa. Both derive from putative ligand-binding alpha 3 subunits, with the smaller phosphorylated species probably representing a degradation product of the larger. The increase in receptor phosphorylation caused by incubating the neurons with a cAMP analog parallels the increase observed in the ACh response, with respect to both time course and relative extent. The phosphorylation of ciliary ganglion AChRs differs from that reported for electric organ AChRs, which occurs primarily on the non-ligand- binding gamma and delta subunits and increases the rate of agonist- induced receptor desensitization.

Chronic Nicotine Treatment Up-regulates Human α3β2 but Not α3β4 Acetylcholine Receptors Stably Transfected in Human Embryonic Kidney Cells

“…The largest currents for these cells were between 1 and 1.5 nA. The EC 50 values for activation by ACh or nicotine were 209 Ϯ 26 and 70 Ϯ 6 M, respectively. The Hill coefficients were 1.7 and 1.3, respectively.…”

“…The subunit combination ␣3␤4 gave the most robust responses, typically reaching maximal currents of 1-5 nA, with some cells responding with currents as large as 15-20 nA. The EC 50 values for activation by ACh and nicotine for ␣3␤4 AChRs were 79 Ϯ 8 and 56 Ϯ 15 M, respectively. The Hill coefficients were 1.5 and 1.6, respectively.…”

“…The need to expose the ␤2-containing cell lines to nicotine for functional studies raises the concern that the functional properties of the AChRs might be altered by nicotine exposure. To address this concern we performed a parallel series of experiments in oocytes expressing ␣3␤2 and ␣3␤4 AChRs, which demonstrated that overnight incubation of the oocytes in 100 M nicotine had no substantial effect on the rates of desensitization (which is also more rapid for ␣3␤2 AChRs expressed in oocytes (16,49)), EC 50 values for activation of the ␣3␤2 or ␣3␤4 AChRs by ACh and nicotine, or the efficacy of nicotine (which also is a partial agonist for ␣3␤2 AChRs expressed in oocytes (16,49)) (data not shown).…”

Desensitization of neuronal nicotinic receptors

“…The major consensus phosphorylation sequences on nAChR subunits lie within the large intracellular loop between TM domains 3 and 4 (Swope et al, 1992). Several of these sites have been shown to be directly phosphorylated in vitro by PKA or PKC (Vijayaraghavan et al, 1990;Nakayama et al, 1993;Moss et al, 1996;Wecker et al, 2001), and may underlie the observed phosphorylation of certain nAChRs in vivo (Viseshakul et al, 1998). In particular, serine 368 on the ␣4 subunit, may underlie the enhanced recovery from desensitization of oocyteexpressed ␣4␤2 nAChRs mediated by Ca 2ϩ /PKC, because mutation of this residue limits recovery (Fenster et al, 1999a) and, although it has a higher affinity for PKA, serine 368 is a substrate for PKC (Wecker et al, 2001).…”

Regulatory mechanisms that govern nicotinic synapse formation in neurons

“…Treatment of CG neurons with VIP increases ACh responses, but not nAChR surface levels, by increasing intracellular cAMP (Gurantz et al, 1994). Elevated cAMP levels enhance ACh responses by converting existing surface nAChRs from a nonfunctional "silent" state to a functional state via posttranslational mechanisms (Margiotta et al, 1987;Vijayaraghavan et al, 1990). Further, CG neurons express the receptors for a second potential input-derived factor, pituitary adenylate cyclase-activating polypeptide (PACAP), a close relative of VIP.…”