Down-regulation of glycine receptor channels by protein kinase C in Xenopus oocytes injected with synthetic RNA

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 24 (1994) 295-300

Research Report

Down-regulation of glycine receptor channels by protein kinase C in Xenopus oocytes injected with synthetic RNA Mutsumi Uchiyama a, Keiko Hirai c, Fumio Hishinuma c, Hiroyuki Akagi h,. "Departments of Anesthesiology and Resuscitology, b Pharmacology, Gunma University School of Medicine, Showa-machi, Maebashi, Gunma 371, Japan c Molecular Biology, Mitsubishi Kasei Institute of Life Sciences, Tokyo 194, Japan

(Accepted 1 March 1994)

Abstract

Interaction of protein kinase C (PKC) with glycine receptor channels was examined using Xenopus oocytes expressing homomeric a l glycine channels. 4/3-Phorbol 12-myristate 13-acetate (4/3-PMA), an activator of PKC, reduced the response to glycine; this effect was inhibited in the presence of staurosporine, a PKC inhibitor. By contrast, 4a-PMA, a poor PKC stimulant, did not affect the glycine currents. Thus, the PKC system is involved in negative-regulation of the glycine receptor channels. The results obtained from experiments with mutant receptors suggest that phosphorylation of the intracellular serine residue at 419 may relate to modification of the channel function. Key words: Ligand-gated channel; Glycine receptor; Neurotransmitter; Phosphorylation; Phorbol ester; Point mutation

I. Introduction

Inhibitory neurotransmission of fast time course is mainly mediated by G A B A and glycine, which gate anion-selective channels differing in physiological and pharmacological properties [4,5]. The receptor channels have oligomeric (presumably pentameric) complexes consisting of subunit proteins [40]. Molecular cloning works have disclosed that various types of receptor subunits exist in the central nervous system, although their expression patterns vary with the type of tissue and the age of animal. For the glycine receptor channels, so far three a ( a l , a2, and a3) and one /3 subunits have been identified [1,8,9,10,18,19]. Among these subunits, the a subunits seem to play a substantial role in receptor-channel function, because they possess domains associated with ligand binding and channel formation. X e n o p u s oocytes injected with single R N A encoding each a subunit can generate membrane currents in response to glycine, and can b e depressed by strychnine, a specific blocker of glycine

* Corresponding author. Fax: (81) 272-20-7961. E-mail: hakagi@sb. gunma-u.ac.jp, 0169-328x/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0169-328X(94)00037-F

receptor channels [1,2,10,18,19,28,34]. In outside-out patches excised from the oocyte membrane, moreover, activity of the glycine-gated single channel can be observed [38]. Therefore, it is likely that the glycine receptors expressed in oocytes are comprised of a homo-oligomeric assembly of the a subunit and are functional ion channels. Thus, the homomeric a channels expressed in oocytes are useful for determining domains related to the receptor-channel functions because of their structural simplicity. It is generally accepted that ligand-gated channels are directly phosphorylated by a variety of protein kinases. Moreover, physiological and pharmacological studies have demonstrated that channel function is upor down-regulated by receptor phosphorylation [42]. These mechanisms might also be involved in maintenance of neurotransmission and synaptic plasticity [26,37]. It has been shown that the cyclic AMP-dependent protein kinase (PKA) regulates various ligand-gated channels including glycine receptors. For instance, the glycine-gated currents in rat spinal trigeminal neurons are increased after treatment with various PKA activators [33]. Protein kinase C (PKC) is another crucial kinase that can modulate function of various ion channels [23,29]. However, much less is known about physi-

M. Uchiyama et al. / Molecular Brain Research 24 (1994) 295-300

296

ological significance of interaction of PKC with glycine receptor channels, although biochemical data indicating that bovine 48-kDa subunit protein (al) is a substrate of PKC have been demonstrated [27]. The aim of the present study was to determine the mechanism by which PKC regulates homomeric a l glycine receptor channels expressed in oocytes, and to identify the site of phosphorylation.

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The sense (s) RNA encoding the a l glycine receptor subunits (rat) was transcribed in vitro from the plasmid pSPT19 containing the a l complementary (c) DNA [1,38]. After capping at the 5'-end, the sRNA was suspended in sterile distilled water at a concentration of 1 mg/ml (calculated from the OD260 value) and stored at -80°C. Mutant cDNAs of the ~1 glycine receptor were prepared by the polymerase chain reaction (PCR) methods as previously described [13]. The mutant cDNAs were subcloned into pSPT19 and the nucleotide sequence was analyzed. The mutant sRNAs were synthesized in vitro as the wild sRNA. Messenger (m) RNA was extracted from brains of mature Wistar rats by the phenol/chloroform method and partially purified using oligo-dT cellulose chromatography [3,16]. About 50 nl of each RNA solution (0.1 mg/ml for sRNAs, and 1 mg/ml for rat brain mRNA) was injected into oocytes (stage V or VI) of Xenopus laet:is. The oocytes were treated with collagenase (Sigma type I, 0.6-1.0 mg/ml for 1 hr) to remove follicular cell layers. After incubation for 2-5 days at 20°C in modified Barth's solution (for composition see [36]), the oocyte was placed in a 0.5-ml bath chamber, which was perfused continuously with frog Ringer solution at room temperature, and it was voltage-clamped at a holding potential of - 7 0 mV with two glass capillary microelectrodes [11]. All drugs were applied by superfusion. Phorbol esters (Sigma) were dissolved in dimethyl sulfoxide at a concentration of 200 /~M, and they were diluted to 1-100 nM in a frog Ringer solution. Staurosporine (Sigma) was made as a 1 mM stock in dimethyl sulfoxide.

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Fig. 1. Effects of 4/3-PMA on agonist-induced membrane currents in Xenopus oocytes. The oocytes were injected with sense RNA encoding glycine receptor a l subunit (A,B) or messenger RNA isolated from adult rat brain (C). After incubation for 2 to 5 days, the oocytes were superfused with frog Ringer solution and voltage-clamped at - 7 0 mV. A,B: glycine (0.1-0.3 mM) was dissolved in the Ringer solution and applied by superfusion at intervals of 5 to 8 rain. C: kainic acid (0.1 mM) was applied at 8 min intervals. 4/~-PMA (10 to 30 nM) was administrated during the periods indicated by the thick horizontal bars. Downward deflection denotes inward currents. The glycine response behind the line breaks in A was recorded at 35 rain after the removal of 4/3-PMA. In order to measure membrane resistance, brief step pulses to - 9 0 mV for 5 sec were given between responses (marked with * in A and C).

3. R e s u l t s

The voltage-clamped oocytes (Vh - 7 0 mV) injected with sRNA encoding a l subunit produced inward membrane currents in response to the addition of glycine. The responses were in a concentration-dependent manner (50 /zM to 10 mM), and when the agonist at 0.5 mM was applied, the amplitudes were usually larger than 200 nA; while non- and water-injected oocytes did not, or if any, elicited very small amplitude (smaller than 5 nA) of glycine currents. As reported previously, the currents seemed to be generated through newly expressed glycine channels in the oocytes, judging from their physiological and pharmacological properties [1,2]. Figs. 1 and 2 show the effects of 4/3-phorbol 12myristate 13-acetate (4/3-PMA), a potent and persistent stimulator of PKC [6,24], on the glycine currents. As shown in Fig. 1A, B, during the treatment of the oocytes with Ringer solution containing 4/3-PMA (10-

30 nM) for 5 to 10 min, the glycine currents began to reduce, and after removal of the drug, the currents still continued to decrease. The average time required to reach half amplitude of the control responses was 40 min (Fig. 2A), although extent of the depression by 4/3-PMA varied in an individual oocyte (from 20 to 80% of the control responses when measured at a time of 40 min; Fig. 2A). Thus, the pharmacological action of 4/3-PMA occurred with slow time course. This depression of glycine currents was not accompanied by any change in membrane resistance (Fig. 1A,C). One possible explanation for the effect of 4/3-PMA is a nonspecific action such as membrane stabilization, resulting in the hindrance of the channel opening mechanism [14]. In order to check this possibility, effect of 4/3-PMA on the kainic acid-induced currents in oocytes injected with rat brain mRNAs were examined. In accordance with previous reports [31,41], the kainate-

M. Uchiyama et al. / Molecular Brain Research 24 (1994) 295-300

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Time (rain) Fig. 2. Time course of action of 4/3-PMA on the glycine-induced membrane currents. A: effects of fl-PMA (10 nM) on glycine- (o) and kainate- (©) induced currents were compared. B: actions of 4fl-PMA (10 nM, o) and 4a-PMA (100 nM, <3) on the glycine currents were compared. Each point represents the relative amplitude (mean with S.D., n = 3-5) expressed as a percentage of control responses (time 0), and plotted against time. PMAs were added to the superfusion medium during the period indicated by the horizontal bar.

induced currents were not affected by 4/3-PMA at a concentration of 10 nM (Figs. 1C and 2A). Furthermore, 4a-PMA, an inactive analog for PKC [6], caused no detectable change in amplitude of the glycine currents (Fig. 2B). Therefore, it is likely that the depression of glycine currents resulted from the activation of

PKC in oocytes. To confirm this, we examined the effect of staurosporine, a potent inhibitor of PKC [39]. While the action of 4fl-PMA on the glycine currents was observed in absence of staurosporine (76 _+7.2% of control response, n = 4, Fig. 3A), treatment of oocytes with the PKC inhibitor (1 /zM) resulted in abolishing the action of 4fl-PMA on the glycine currents (103 + 2.2%, n = 3, Fig. 3B). These results strongly suggest that homomeric a l glycine channels are down-regulated by PKC. The next question in the present study is how PKC modifies the glycine channels. Does PKC directly phosphorylate the glycine receptor subunit, or does PKC phosphorylate another substance which modulates channel activity? To answer this question, we constructed mutant cDNAs. It is proposed that, in many ionotropic receptor channels, there exist phosphorylation residues in the intracellular loop which is flanked

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M. Uchiyama et al. /Molecular Brain Research 24 (1994) 295-300

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by membrane-spanning domains 3 and 4 [37]. In the a l glycine receptor channels, this region is assumed to consist of 80 amino acids in which 7 s e r i n e / t h r e o n i n e residues are included [1,2,9]. It is probable that one or more of these amino acids are candidates for phosphorylation. Two mutant DNAs used in the study are indicated in Fig. 4. One of the mutated receptor subunits is M38, in which alanine is substituted for serine at position 419 (for the position number see [1]), and the other is M44, in which all of s e r i n e / t h r e o n i n e residues except serine at 419 are replaced with alanine. The oocytes injected with sRNA transcribed from M38 or M44 DNAs could generate currents (20-200 nA in amplitude) in response to glycine (1 mM). As shown in Fig. 5A, the glycine currents generated through the M44 receptor were also depressed by 4/3-PMA (10-30 nM) with a slow time course. The average time to reach half amplitude was 30 min; this is as long as observed with the wild-type receptor (see Fig. 2A). Thus, it seems to be obvious that the mutant M44 receptor is sensitive to 4/3-PMA, suggesting that 6 s e r i n e / t h r e o n i n e residues (Fig. 4) are not related to the 4/3-PMA-action. By contrast, the effect of 4/3-PMA on the glycine currents through M38 receptor is somewhat unclear. Between the examined oocytes expressing M38 receptor, some ones elicited glycine currents with a constant amplitude, and in these oocytes, 4/3PMA did not, or if any, weakly affected the glycine currents. Three examples are shown in Fig. 5B-1. However, it was very difficult to evaluate the action of 4/3-PMA on M38 receptor in many other oocytes, because currents generated through the receptor were 'run-down'; that is to say, the currents decreased by repeated application of glycine, even before the exposure of the oocytes with 4/3-PMA. Examples obtained in 3 different oocytes are illustrated in Fig. 5B-2. Nevertheless, when the plotted time courses of declining currents were compared between before and after treatments with 4/3-PMA, the apparent slope values do not seem to differ considerably.

4. D i s c u s s i o n

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Time (rain.) Fig. 5. Effect of 4/3-PMA on glycine-evoked membrane currents generated through the mutated receptor channels, M44 (A) and M38 (B). Glycine (1 raM) was applied as described before. A: each point represent relative amplitude in average to control responses as 100%. 4/3-PMA (10 nM) was perfused during the period indicated by the horizontal bar. B-l: examples obtained from 3 individual oocytes (©, e, • ) , which generated glycine currents with constant amplitude, and exhibited poor sensitivity to 4/3-PMA, are demonstrated. B-2" sample records obtained from 3 other oocytes (©, e, • ) in which the M38 receptor could not maintained glycine currents before applying 4/3-PMA.

Modulatory effects of protein kinases on vertebrate neuronal signaling are of particular interest because they may be relevant to synaptic plasticity [30,37]. Many ligand-gated channels are targets of the PKC systems; specifically, phosphorylation of nicotinic ACh, G A B A A and glutamate (NMDA) receptors by PKC cause modulation of their functions [15,17,20,22,31,32]. Glycine receptor channels are also a likely target of PKC. Ruiz-Gomez et al. have provided biochemical evidence that 48 kDa protein ( a l subunit) is a substrate of PKC in vitro [27]. In the present study, using Xenopus oocytes expressing functional cloned glycine receptors

M. Uchiyama et al. /Molecular Brain Research 24 (1994) 295-300

(homomeric a l channels), we found that 4/3-PMA suppressed the glycine-induced currents. We presumed that this effect is due to the activation of PKC in oocytes by following reasons: (1) the effective concentration of 4/3-PMA was as low as 1 to 30 nM. These concentrations seem to be reasonable judging from other biochemical studies [6,23]; (2) 4a-PMA, which does not activate PKC, did not affect the glycine currents even at a high concentration of 100 nM; and (3) staurosporine, a potent inhibitor of PKC, blocked the action of 4/3-PMA on glycine currents. How does phosphorylation by PKC reduce glycine receptor channel activity? A likely mechanism is that PKC acts by directly phosphorylating a component of the glycine receptor channel. Several acceptor sites (serine/threonine residues) for phosphorylation are present in the intracellular domain flanked by TM3 and TM4 of the a l subunit of glycine receptor, and in fact, biochemical data by Ruiz-G6mez et al. indicate that the serine residue at position 419 is the specific phosphorylation site for PKC in the o~1 subunit [27]. Moreover, the sequence of amino acids around the serine at 419 includes a consensus motif ( K / R X S / T ) for PKC-phosphorylation [25]. If the phosphorylation of this site contributes to the down-regulation of the glycine current, 4/3-PMA should not affect the currents generated through a mutated receptor in which the putative phosphorylation site for PKC is blocked. This possibility was evaluated using mutated receptors, M44 and M38 (Fig. 4). The obtained results from the experiments using M44 receptor demonstrated that serine/ threonine residues except the serine at 419 do not contribute to the down-regulation of the glycine receptor channels by PKC. This result is compatible with presumed functional importance of the serine at 419 with respect to the PKC-induced down-regulation. This is also supported by a piece of data from the experiment with M38 receptor in which glycine currents recorded from some oocytes were insensitive or, at least, less sensitive to 4/3-PMA (Fig. 5B-l). Nevertheless, the definite conclusion for a role of the serine at 419 as the target of PKC should be reserved, because, the glycine currents through M38 receptor in many other oocytes were accompanied by 'unexpected behavior'; specifically, spontaneous run-down of the response was observed (Fig. 5B-2, discussed later). Under these current recordings, it is troublesome to appraise effects of 4/3-PMA. Further experiments will be required to obtain more clear-cut evidence for the role of serine 419 in the PKC-induced down-regulation mechanism. Apart from this problem, M38 receptor may provide a clue for solving spontaneous run-down mechanism. Most of the oocytes injected with M38 sRNA generated membrane currents of large amplitude in response to pulse-applications of glycine when the begin-

299

ning of recordings, but by repeating the pulse at longer intervals of 10 rain, the currents continue to decrease in amplitude. This contrasts with the glycine currents generated through the wild o~1 and the mutated M44 receptor, which generally maintain its amplitude of currents. It has been reported for the case of GABA A and NMDA receptor channels that phosphorylation of the channel subunits is important for maintenance of the currents generation [7,12,21,35]. A similar mechanism might be involved in the glycine receptor channels, and the serine residue at 419 is responsible for the mechanism.

Acknowledgements We are grateful to F. Ozawa and T. Miyazaki for their technical assistance, and to Profs. K. Kohama and T. Fujita, Drs. K. Tanaka and H. Shimada for discussion, technical advises and encouragement through this work. This work was supported in part by Ministry of Education, Science and Culture of Japan and Kowa Life Science Foundation.

References [1] Akagi, H., Hirai, K. and Hishinuma, F., Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development, FEBS Lett., 281 (1991) 160-166. [2] Akagi, H., Hirai, K. and Hishinuma, F., Functional properties of strychnine-sensitive glycine receptors expressed in Xenopus oocytes injected with a single mRNA, Neurosci. Res., 11 (1991) 28-40. [3] Akagi, H. and Miledi, R., Expression of glycine and other amino acid receptors by rat spinal cord mRNA in Xenopus oocytes, Neurosci. Lett., 95 (1988) 262-268. [4] Betz, H., Ligand-gated ion channels in the brain: The amino acid receptor superfamily, Neuron, 5 (1990) 383-392. [5] Bormann, J., Hamill, O.P. and Sakmann, B., Mechanism of anion permeation through channels gated by glycine and 3'aminobutyric acid in mouse cultured spinal neurones, J. Physiol., 385 (1987) 243-286. [6] Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and Nishizuka, Y., Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters, J. Biol. Chem., 257 (1982) 7847-7851. [7] Chert, Q.X., Stelzer, A., Kay, A.R. and Wong, R.K.S., GABA A receptor function is regulated by phosphorylation in acutely dissociated guinea-pig hippocampal neurones, J. Physiol., 420 (1990) 207-221. [8] Grenningloh, G., Pribilla, I., Prior, P., Multhaup, G., Beyreuther, K., Taleb, O. and Betz, H., Cloning and expression of the 58 kd /3 subunit of the inhibitory glycine receptor, Neuron, 4 (1990) 963-970. [9] Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E.D. and Betz, H., The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors, Nature, 328 (1987) 215-220. [10] Grenningloh, G., Schmieden, V., Schofield, P.R., Seeburg, P.H.,

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Siddique, T., Mohandas, T.K., Becker, C.-M. and Betz, H., Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes, EMBO J., 9 (1990) 771-776. [11] Gundersen, C.B., Miledi, R. and Parker, I., Properties of human brain glycine receptors expressed in Xenopus oocytes, Proc. R. Soc. Lond., B221 (1984)235-244. [12] Gyenes, M., Farrant, M. and Farb, D.H., 'Run-down' of 3'aminobutyric acid A receptor function during whole-cell recording: a possible role for phosphorylation, Mol. Pharmacol., 34 (1988) 719-723. [13] Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. and Pease, L.R., Site-directed mutagenesis by overlap extension using the polymerase chain reaction, Gene, 77 (1989) 51-59. [14] Hockberger, P., Toselli, M., Swandulla, D. and Lux, H.D., A diacylglycerol analogue reduces neuronal calcium currents independently of protein kinase C activation, Nature, 338 (1989) 340-342. [15] Huganir, R.L. and Miles, K., Protein phosphorylation of nicotinic acetylcholine receptors, Crit. Rev. Biochem. Mol. Biol., 24 (1989) 183-215. [16] Ishida, M., Akagi, H., Shimamoto, K., Ohfune, Y. and Shinozaki, H., A potent metabotropic glutamate receptor agonist: electrophysiological actions of a conformationally restricted glutamate analogue in the rat spinal cord and Xenopus oocytes, Brain Res., 537 (1990) 311-314. [17] Kelso, S.R., Nelson, T.E. and Leonard, J.P., Protein kinase C-mediated enhancement of NMDA currents by metabotropic glutamate receptors in Xenopus oocytes, J. Physiol., 449 (1992) 705-718. [18] Kuhse, J., Schmieden, V. and Betz, H., Identification and functional expression of a novel ligand binding subunit of the inhibitory glycine receptor, J. Biol. Chem., 265 (1990) 22317-22320. [19] Kuhse, J., Schmieden, V. and Betz, H., A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit, Neuron, 5 (1990) 867-873. [20] Kutsuwada, T., Kashiwabuchi, N., Mori, H., Sakimura, K., Kushiya, E., Araki, K., Meguro, H., Masaki, H., Kumanishi, T., Arakawa, M. and Mishina, M., Molecular diversity of the NMDA receptor channel, Nature, 358 (1992) 36-41. [21] MacDonald, J.F., Mody, I. and Salter, M.W., Regulation of N-methyl-D-aspartate receptors revealed by intracellular dialysis of murine neurones in culture, J. PhysioL, 414 (1989) 17-34. [22] Miles, K. and Huganir, R.L., Regulation of nicotinic acetylcholine receptors by protein phosphorylation, Mol. Neurobiol., 2 (1988) 91-124. [23] Nishizuka, Y., Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Science, 258 (1992) 607-614. [24] Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D. and Ullrich, A., The complete primary structure of protein kinase C - - the major phorbol ester receptor, Science, 233 (1986) 853-859. [25] Pearson, R.B. and Kemp, B.E., Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations, Methods Enzymol., 200 (1991) 62-81.

[26] Raymond, L.A., Blackstone, C.D. and Huganir, R.L., Phosphorylation of amino acid neurotransmitter receptors in synaptic plasticity, Trends Neurosci., 16 (1993)147-153. [27] Ruiz-G6mez, A., Vaello, M.L., Valdivieso, F. and Mayor, F. Jr., Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C, J. BioL Chem., 266 (1991) 559-566. [28] Schmieden, V., Grenningloh, G., Schofield, P.R. and Betz, H., Functional expression in Xenopus oocytes of the strychnine binding 48 kd subunit of the glycine receptor, EMBO J., 8 (1989) 695-700. [29] Shearman, M.S., Sekiguchi, K. and Nishizuka, Y., Modulation of ion channel activity: a key function of the protein kinase C enzyme family, Pharmacol. Rev., 41 (1989) 211-237. [30] Sibley, D.R., Benovic, J.L., Caron, M.G. and Lefkowitz, R.J., Regulation of transmembrane signaling by receptor phosphorylation, Cell, 48 (1987) 913-922. [31] Sigel, E. and Baur, R., Activation of protein kinase C differentially modulates neuronal Na +, Ca 2+, and y-aminobutyrate type A channels, Proc. Natl. Acad. Sci. USA, 85 (1988) 6192-6196. [32] Sigel, E., Baur, R. and Malherbe, P., Activation of protein kinase C results in down-modulation of different recombinant GABAA-channels, FEBS Lett., 291 (1991) 150-152. [33] Song, Y. and Huang, L.-Y.M., Modulation of glycine receptor chloride channels by cAMP-dependent protein kinase in spinal trigeminal neurons, Nature, 348 (1990) 242-245. [34] Sontheimer, H., Becker, C.-M., Pritchett, D.B., Schofield, P.R., Grenningloh, G., Kettenmann, H., Betz, H. and Seeburg, P.H., Functional chloride channels by mammalian cell expression of rat glycine receptor subunit, Neuron, 2 (1989) 1491-1497. [35] Stelzer, A., Intracellular regulation of GABAA-receptor function. In T. Narahashi (Ed.) Ion Channels, Vol. 3, Plenum, New York, 1992, pp. 83-136. [36] Sumikawa, K., Parker, 1. and Miledi, R., Expression of Neurotransmitter receptors and voltage-activated channels from brain mRNA in Xenopus oocytes. In P.M. Conn (Ed.), Methods in Neuroscience, Vol. 1, Academic Press, San Diego, 1989, pp. 30-45. [37] Swope, S.L., Moss, S.J., Blackstone, C.D. and Huganir, R.L., Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity, FASEB J., 6 (1992) 2514-2523. [38] Takahashi, T., Momiyama, A., Hirai, K., Hishinuma, F. and Akagi, H., Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels, Neuron, 9 (1992) 1155-1161. [39] Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M. and Tomita, F., Staurosporine, a potent inhibitor of phospholipid/Ca 2÷ dependent protein kinase, Biochem. Biophys. Res. Commun., 135 (1986) 397-402. [40] Unwin, N., The structure of ion channels in membranes of excitable cells, Neuron, 3 (1989) 665-676. [41] Urushihara, H., Tohda, M. and Nomura, Y., Selective potentiation of N-methyl-D-aspartate-induced current by protein kinase C in Xenopus oocytes injected with rat brain RNA, J. Biol. Chem., 267 (1992) 11697-11700. [42] Walaas, S.I. and Greengard, P., Protein phosphorylation and neuronal function, Pharmacol. Rev., 43 (1991) 299-349.