7739-7743, November 1987. Neurobiology. Activators of protein kinase C enhance acetylcholine receptor desensitization in sympathetic ganglion neurons.
Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7739-7743, November 1987 Neurobiology
Activators of protein kinase C enhance acetylcholine receptor desensitization in sympathetic ganglion neurons (neuronal nicotinic acetylcholine receptor/modulation)
JAMES E. G. DOWNING AND LORNA W. ROLE Department of Anatomy and Cell Biology and The Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, New York, NY 10032
Communicated by Charles F. Stevens, June 24, 1987
Recent studies suggest that phosphorylation ABSTRACT may regulate the rate of desensitization of nicotinic acetylcholine (AcCho) receptors (AcChoR) in vertebrate muscle and Torpedo. It is not known if phosphorylation is involved in regulation of the neuronal AcChoR, however. In this study we examine the possibility that protein kinase C might regulate nicotinic AcChoR function in neurons. Several activators of protein kinase C (1-oleyol-2-acetylglycerol, phorbol 12,13diacetate, and phorbol 12,13-dibutyrate) were tested for their ability to modulate AcChoR function in embryonic chicken sympathetic ganglion neurons. Neurons were voltage-clamped at the resting potential, and the response to AcCho was tested before and after treatment with activators of protein kinase C. We find that all of these agents enhance the rate of decay of AcCho-induced current without affecting peak current amplitude or cellular input resistance. The drugs were ineffective if applied concurrently with AcCho: significant effects could be detected after 60 sec of pretreatment. A phorbol that does not increase protein kinase C activity (48-phorbol) was ineffective in enhancing the decay of AcCho-induced current. Thus, the effects of these agents on AcChoR function are likely to be mediated by their interaction with C kinase, rather than by direct interaction with the AcChoR channel. Our data suggest that kinase C may regulate agonist-induced desensitization of the neuronal AcChoR channel.
Many studies indicate that protein phosphorylation plays a central role in the modulation of neuronal excitability (see refs. 1-5 for reviews). Protein kinase A is implicated in the modulation of several voltage-gated conductances (see refs. 1, 4, and 6 for reviews; refs. 7-9). The Ca2+- and phospholipid-dependent kinase, protein kinase C (10-12), is also involved in the modulation of an array of voltage-dependent channels (13-19), including an enhancement of Ca2+ current in Aplysia bag cell neurons (17), a decrease in Ca2+ current in dorsal root ganglion neurons (15), and modulation of Cland a variety of K+ conductances in several systems (refs. 13 and 14; see ref. 16 for a review). In these studies, the function of the voltage-gated conductances is altered by drugs that enhance kinase activity (1, 15-17) or by the kinase itself (see ref. 4 for a review; refs. 9, 17, and 19). In some cases, correlative biochemical studies have demonstrated neurotransmitter modulation of kinase activity (see refs. 1 and 4 for reviews); yet in all these studies, it is not known ifthe channel itself is the phosphoprotein. Protein phosphorylation also may be involved in the modulation of chemically gated channels. It has been known for some time that the nicotinic acetylcholine (AcCho) receptor (AcChoR) of Torpedo is directly phosphorylated (20, 21) by several different kinases (refs. 22-24; see ref. 25 for a review). Recent evidence now suggests that kinaseThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ยง1734 solely to indicate this fact.
mediated phosphorylation of muscle and electroplax nicotinic AcChoRs enhances the rate of agonist-induced desensitization (26-31). The physiological stimulus for kinase activation and the resultant desensitization in these systems is unknown, however. Studies of neuronal AcChoRs indicate that peptides, such as substance P, that are present at certain cholinergic synapses can modulate nicotinic AcChoR desensitization. Substance P enhances the rate of AcCho-induced desensitization in chromaffin cells (32-35) and in both sympathetic and parasympathetic neurons (36, 37). In view of the data discussed above demonstrating that phosphorylation enhances muscle AcChoR desensitization, it is possible that substance P modulates neuronal AcChoR desensitization via protein phosphorylation. We have examined the possibility that phosphorylation regulates neuronal AcChoR desensitization by measuring AcCho-induced currents in voltage-clamped sympathetic ganglion neurons. The rate of decay of macroscopic currents evoked by agonist was determined before and after treatment of neurons with activators of protein kinase C. Our studies indicate that the rate of receptor desensitization is greatly enhanced by several agents that activate protein kinase C. These findings suggest that the modulation of neuronal nicotinic AcChoR function may involve changes in protein kinase C-mediated phosphorylation.
MATERIALS AND METHODS Cell Culture. Sympathetic ganglion neuronal cultures were prepared as described (36) and plated on polyornithinecoated tissue culture dishes in Dulbecco's modified Eagle's medium supplemented with horse serum (10%), penicillin (50 units per ml), streptomycin (50 ,ug/ml), glutamine (2 mM), chicken embryo extract (5%), and nerve growth factor (0.1 ,ug/ml; 2.5 S, kind gift of P. Osborne, Washington University Medical School, St. Louis). These growth conditions suppress the proliferation of nonneuronal cells resulting in cultures comprised of '90% cells with neuronal morphology that are sensitive to applied AcCho (see below) and contain catecholamine as assessed by histofluorescence techniques (L.W.R., unpublished data). Recording and Data Analysis. Macroscopic currents were recorded with the whole-cell variation of the patch-clamp method (38). The solution in the patch pipette contained 140 mM KCl, 2 mM MgCl2, 11 mM EGTA, 1 mM CaC12, 10 mM Hepes, 5 mM MgATP, pH 7.2. The recording/bathing medium contained 140 mM NaCI, 6 mM KCl, 0.8 mM MgCl2, 10 mM CaCI2, 12.5 mM Hepes, and 5 mM glucose, pH 7.4. Under these recording conditions, the neurons respond to nicotinic agonists but not to muscarinic agonists and maintain Abbreviations: AcCho, acetylcholine; AcChoR, acetylcholine receptor; OleAcGro, 1-oleoyl-2-acetylglycerol; P(OAc)2, phorbol
12,13-diacetate; P(OBt)2, phorbol 12,13-dibutyrate. 7739
7740
Neurobiology: Downing and Role
stable rest potentials for at least 5 hr. Cells were voltageclamped to the rest potential (-60 mV) unless otherwise stated. The macroscopic current evoked by AcCho in the presence or absence of test agents was stored on either FM tape (Racal, Vienna, VA) or videotape with a PCM digitizer (Neuro Data, DR 384, New York) for subsequent analysis. The rate of decay of the evoked current was computed two ways. In some experiments the decays were fit by using an analysis routine written and kindly provided by S. Schuetze (Columbia University) and were run on a DEC 11/73-based microcomputer. The decay-time course typically was fit by the sum of two exponential curves by an iterative nonlinear leastsquares regression routine. In addition, we have used a simpler measure of current decay: the comparison of the current level 5 sec after the onset of agonist application (I5) with the peak of the evoked current (Ip). In this analysis, the percentage decay from peak is measured as 100[1 - (I5/Ip)], mean + SEM, and is referred to throughout the text as Id5. Comparison of the two approaches indicated that they gave qualitatively the same results (see Tables 1 and 2). Statistical significance was evaluated by using a two-tailed Student t test
A
RESULTS AcCho-Induced Current Decay Is Enhanced by OleAcGro. Fig. 1A shows a sample response of a sympathetic neuron voltage-clamped to the resting potential and exposed to 20 ,uM AcCho. The inward current rises steeply and then decays despite the continued presence of agonist. The observed decay is dependent on agonist concentration, with maximal decay of the current observed at 50 ,uM AcCho, half-maximal decay at concentrations of -12 ,uM, and no detectable decay during a 10-second application of 1 MuM AcCho or less (unpublished data). The observed decay of the AcChoinduced current is presumably the result of agonist-induced desensitization. The decay of the AcCho-induced current is described well by the sum of two exponential curves with relatively fast (Tf) and slow (r) time constants of decay. The record shown in Fig. 1B displays the agonist-induced current evoked by AcCho in the presence of the protein kinase C activator, OleAcGro. This record was obtained from the same neuron as in Fig. 1A after a 3-min pretreatment with a maximally effective concentration of OleAcGro (85 ,uM; see below). The decay of the AcCho-induced current after OleAcGro treatment was also comprised of two components,
but T. decreased from 29.0 sec to 7.6 sec, and the relative contribution of this component to the overall decay (%S)
1.6 sec
Tr = Ts % K
=
29.0 sec
=
67.9
B
Tf = Ts
(39).
Drug Preparation and Application. Drugs were administered by pressure application, at known concentrations, from blunt-tipped (2-5 tum) pipettes positioned -20 tum from the cell soma. Control experiments indicate that drugs applied in this manner are diluted by
