and may play an important role in synaptic plasticity. -Swope,. S. L.; Moss, S.J.; Blackstone,. C. D.; Huganir,. R. L. Phosphorylation of ligand-gated ion channels:.
IViV.$: of ligand-gated
Phosphorylation mode
of synaptic
SHERIDAN
L
SWOPE,
ion hauneIs:
plasticity STEPHEN
‘5.
MOSS,
Department of Neuroscience Howard Hughes Baltimore, Maryland 21205, USA
a possible
CRMG
M die .
L *LiMIKStONE, AND RICHA*D L HUGANIR1 sdtvicThuhns Hopksns Unwemlty School of Medicme :.
#{149}.
ABSTRACT Most neurotransmitter receptors examined to date have been shown either to be regulated by protein phosphorylation or to contain consensus sequences for
kinases. The reverse reaction, group from the phosphoprotein, phoprotein phosphatases (2).
phosphorylation by protein kinases. Neurotransmitter ceptors that mediate rapid synaptic transmission in nervous system are the ligand-gated ion channels and clude the nicotinic acetylcholine receptors of muscle
calmodulin-dependent
rethe
inand
nerve and the excitatory and inhibitory amino acid receptors: the glutamate, GABAA, and glycine receptors. These receptors are multimeric proteins composed of
homologous subunits which each span the membrane several times and contain a large intracellular loop that is a mosaic of consensus sites for protein phosphorylation. Recent evidence has suggested that extracellular signals released from the presynaptic neuron, such as neurotransmitters and neuropeptides as well as an extracellular matrix protein, regulate the phosphorylation of ligandgated ion channels. The functional effects of phosphorylation are varied and include the regulation of receptor desensitization rate, subunit assembly, and receptor aggregation at the synapse. These results suggest that phosphorylation of neurotransmitter receptors represents a major mechanism in the regulation of their function and may play an important role in synaptic plasticity. -Swope, S. L.; Moss, S.J.; Blackstone, C. D.; Huganir, R. L. Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity. FASEB J 6: 25142523; 1992. Key Words: protein kinases receptors ion channels
signal transduction
neurotransmitter
RECEPTORS MEDIATE signal transduction the postsynaptic membrane of synaptic connections between cells in both the central and peripheral nervous systems. This pivotal role in the mechanism of synaptic transmission suggests that neurotransmitter receptors may be potential targets at which synaptic plasticity could occur. Modulation of the function, expression, or density of neurotransmitter receptors in the postsynaptic membrane could have profound effects on the efficacy of synaptic transmission. A variety of data now suggest that protein phosphorylation of neurotransmitter receptors is a primary mechanism for the regulation of neurotransmitter function and may play a major role in the regulation of synaptic transmission. Protein phosphorylation is widely recognized as one of the primary modes of regulation of cellular processes (1, 2). Phosphorylation is a process by which the highly charged phosphate group of ATP is transferred to a serine, threonine, or tyrosine residue of a substrate protein, thus altering its functional properties (1). This covalent modification of the substrate protein is catalyzed by enzymes known as protein NEUROTRANSMITTER
at
2514
hydrolysis of the phosphate is catalyzed by the phosThe most familiar serine/ threonine-specific protein kinases are those regulated by intracellular second messengers: cAMP-dependent protein kinases (PKA),2 cGMP-dependent protein kinases, the calcium!
protein
kinases (CAM-K),
and the
phospholipid/calcium-dependent protein kinases or protein kinase C (PKC) (1). In addition to these second messengerregulated protein kinases there are many second messengerindependent serine/threonine protein kinases as well as a unique class of protein kinases that exclusively phosphorylate tyrosine’residues in their substrates (3). The highest levels of expression for many protein kinases occur in the central nervous system, suggesting their importance in the regulation of neuronal function. Neurotransmitter receptors can be classified into two broad categories: the ligand-gated ion channels and guanine nucleotide binding protein (G protein) linked receptors. These two classes of receptors differ in their oligomeric structure and transmembrane topology as well as in the molecular mechanisms by which they transduce their signals. This review focuses on the modulation of ligand-gated ion channels by protein phosphorylation; the regulation of G protein-linked receptors by protein phosphorylation has recently been reviewed elsewhere (4, 5). Ligand-gated ion channels mediate rapid excitatory and inhibitory synaptic transmission in the nervous system and include the muscle nicotinic acetylcholine receptor (AChR), the neuronal AChR (nAChR), the 7-amino butyric acid (GABAA) receptor, the glycine receptor, the Nmethyl-D-aspartate (NMDA) receptor, and the a-amino-3hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor. Binding of ligand to these receptors induces the rapid opening of their ion channels, which are selectively permeable to either cations or anions, thereby altering the membrane potential of the postsynaptic cell. The members of the ligandgated ion channel class of neurotransmitter receptors are pentameric complexes of subunits which are integral membrane proteins arranged around a central aqueous pore (Fig. l#{192}).
‘To whom correspondence should be addressed, at: Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 900 PCTB, Baltimore, MD
21205-2185, USA. 2Abbreviations: PKA, cAMP-dependent protein kinase; PKC, protein kinase C; AChR, nicotinic acetylcholine receptor; nAChR, neuronal AChR; GABA, gamma amino butyric acid; NMDA, Nmethyl D-aspartic acid; ACh, acetylcholine; CGRP, calcitonin gene-related peptide; bFGF, basic fibroblast growth factor; VIP, vasoactive intestinal peptide; CAM-K, calcium/calmodulin-dependent protein kinase; LTP, long-term potentiation; PTK, protein tyrosine kinases; CNS, central nervous system.
0892-663812/0006-2514101.50.
© FASEB
mitter receptors for which sequence information is available. Many subunits of the ligand-gated ion channels contain these protein kinase recognition motifs in the major intracellular loop between transmembrane domains M3 and M4, suggesting that protein phosphorylation of these subunits may Occur in vivo. In this review, we discuss studies that support a role for protein phosphorylation in the regulation of ligand-gated ion channels.
A
THE NICOTINIC MUSCLE AND
B
P-gmC
1. Schematic model of the topology of an idealized ligandgated ion channel. A) Pentameric subunit structure viewed through the plane of the membrane. B) Membrane topology viewed through the plane of the membrane of an individual subunit containing transmembrane domains M1-M4. P indicates the region of the subunit that contains the phosphorylation sites for the various protein kinases. C) Schematic model representing the arrangement of the transmembrane a-helices within each subunit around the central ion channel. Figure
These subunits are structurally homologous to one another, being composed of a large extracellular NH2-terminal region and four putative transmembrane domains (M1-M4), as shown in Fig. lB and Fig. 1C. Many recent biochemical studies have demonstrated that ligand-gated ion channels are directly phosphorylated by a variety of protein kinases. In addition, physiological studies using drugs, neurotransmitters, hormones, and neuropeptides to modulate intracellular protein kinases have provided evidence that phosphorylation regulates the function of ligand-gated ion channels. Moreover, determination of the primary sequence requirements for the recognition of substrates by the various protein kinases (6) has made it possible to predict potential sites of phosphorylation in neurotrans-
RECEPTORPHOSPHORYLATION
ACETYLCHOLINE ELECTRIC ORGAN
RECEPTOR
OF
The AChR is the ligand-gated ion channel found at the neuromuscular junction which mediates depolarization of the postsynaptic membrane upon binding acetylcholine (ACh). Because of its enrichment in the electric organs of Torpedo calfornica and Electrophorus electricus, the AChR has been extensively characterized, serving as a model for the study of the structure, function, and modulation of ligand-gated ion channels (7). The AChR is a 250-kDa pentameric complex of four types of subunits in a stoichiometry of a2/37h. Each subunit is an integral membrane protein, and the five subunits have been proposed to be arranged around a central aqueous pore (Fig. IA). The receptor has two ACh binding sites, one on each a subunit, which when occupied by ligand triggers the opening of the channel, allowing permeation by cations (7). The structural and functional characteristics of the AChR have been further elucidated by molecular cloning and sequencing. The subunits are highly homologous proteins, each consisting of a large extracellular NH2-terminal region and four putative transmembrane domains (M1-M4), as shown in Fig. lB. The ACh binding site is located on the extracellular NH2terminal domain of the a subunit whereas the channel pore is believed to be lined with a-helices of the M2 membranespanning region of the five subunits (see Fig. 1C)(7). Phosphorylation of the AChR by three different protein kinases endogenous to Torpedo electroplax postsynaptic membranes has been demonstrated, with each kinase showing subunit specificity. PKA phosphorylates the and 6 subunits (8), PKC phosphorylates the 6 subunit (9), (10), and a protein tyrosine kinase phosphorylates the 3, , and 6 subunits (11). Originally, the specific amino acid residues phosphorylated by the various protein kinases were proposed by comparing the protein sequence of the receptor subunits with the known sequence preferences of the three protein kinases (11). Recently, the locations of some of the phosphorylation sites have been determined by sequencing phosphorylated peptide fragments of the AChR, confirming most of the original predictions: PKA phosphorylates the subunit on Ser-353 and the 6 subunit on Ser-361 (12) whereas the protein tyrosine kinase phosphorylates the /3 subunit on Tyr-355, the ‘y subunit on Tyr-364, and the 6 subunit on Tyr-372 (13) (see Table 1). Ser-362 and Ser-377/379 on the 6 subunit appear to be the sites of phosphorylation by PKC as indicated by peptide sequencing, the use of synthetic peptide substrates derived from the primary sequence of the AChR, and the use of anti-peptide antibodies (10, 14). Phosphorylation on Ser-361, Ser-377/379, and Tyr-372 of the 5 subunit is further supported by mass spectrometry analysis (15). These phosphorylation sites are conserved in the adult forms of all species examined, with the exception of those on the mammalian subunits (16). However, the PKA phosphorylation site is conserved in the adult form of the AChR subunit, known as the #{128} subunit, in mammals (16). The effects of phosphorylation have been directly examined using purified preparations of the AChR reconstituted into
2515
TABLE
1. Sequences
of the identified
and proposed phosphorylation
sites for various protein
Receptor/subunit
kinases
on subunits of ligand-gated
ion channel? Protein kinase
Sequence
AChR/
348bI1eSerArgAlaAsnAspGluyPheIleArgLyss59
PTK’
AChRJ-y
t46LysProGlnProArgArgArgSerSerPheGly!le357 359IleLysAlaGluGluIleLeuLys367
PKA PKT
AChR/b
“5LeuLysLeuArgArgSerSerSerValGlyTyr365
PKA
368LysAlaGlnGluyPheAsnIleLysSerArg378
PTK
LeuPheMetLysArgProSerValValLys
PKA
ArgAlaValGluGlyValGlnyIleAlaAspHisLeuLys
PTK
nAChR//33
34LysValSerGlyLysArg352
PKC
nAChR.//34
4t6ArgLeuArgSerSerGlyArgPheArg422
PKC
GABAA-R’/$,
402LysGlyArglleArgArgArgAlaSerGlnLeuLys”3
PKA
GABAA-R/72
360GlnGluArgGluAspAspyrGlyyGluLysLeuAsp372
PTK
GABAA-RiYSL
338LeyLeyArgMetPheSerPheLys346
PKC
GlyR/a,
38AlaLysLyslleAspLyslleSerArg392
PKC
GlyR//3
‘#{176}6ArgAspPheGluLeuSerAsnyAspCysTyrGlyLys4’9
PTK
GIuR6
676AlaPheMetSerSerArgArgGlnSerValLeuVal687
nAChR/a4’
dPhosphorylated amino acids are underlined. bThe exact location of the peptides within the numbers. ‘Abbreviations: GABAA-R, GABAA receptor; GIyR, glycine receptor; PTK, protein tyrosine sequence of nAChR a4 is not presently available. liposomes. The reconstituted receptor displays the functional properties of the AChR found in the native membrane including inactivation in the continued presence of agonist, a process termed desensitization. Rapid kinetic techniques were used to analyze the ion transport properties of nonphosphorylated and PKA-phosphorylated AChR (17). The intial rate of ACh-dependent ion transport is not altered; however, the rapid phase of desensitization is eightfold faster for the phosphorylated AChR. Patch-clamp electrophysiological techniques were used to analyze the effect of tyrosine phosphorylation of the reconstituted AChR (18). The singlechannel conductance and the apparent mean channel open time of the AChR are not affected. However, the rate of the rapid phase of desensitization shows a striking correlation with the stoichiometry of tyrosine phosphotylation. A maximal sevenfold increase in the rate occurs upon tyrosine phosphorylation, similar to the effect of AChR phosphotylation by PKA. Thus, phosphorylation of the purified AChR by either PKA or a protein tyrosine kinase appears to have a common functional effect on the desensitization rate of the AChR. The role of phosphorylation in modulating the AChR has also been examined in cell culture and intact muscle, with the PKA-mediated pathway being the most extensively studied. Forskolin, an activator of adenylate cyclase, and CAMP analogs both increase AChR S subunit phosphorylation in primary cultures of rat muscle cells (19) and in the BC3H1 cell line (20). The effect of activation of the PKAmediated pathway on AChR function has been studied in rat muscle. Treatment of muscle preparations with forskolin or cAMP analogs results in an increased rate of desensitizatioi of the AChR (21-24). However, not all laboratories have observed modulation of AChR desensitization by CAMP analogs (25). Furthermore, interpretation of the results of experiments using forskolin is complicated by the finding that forskolin has effects on AChR function that are not mediated by adenylate cyclase but instead appear to result from a direct local anesthetic-like effect on the AChR (25, 26). However, studies that have controlled for these direct effects support the activation of adenylate cyclase in mediating the
2516
Vol. 6
May 1992
PKA subunits
are as indicated
kinase.
dNumbenng
by
the
inclusive
for the amino
acid
effect of forskolin on the AChR (23). In addition, forskolin’s actions on AChR desensitization (23) and phosphorylation (19) are remarkably similar in time course, concentration dependence, and pharmacology. At the single-channel level, forskolin does not alter either the single-channel conductance or the mean channel open time, but it does decrease the frequency of channel openings (23). Moreover, treatment of muscle cells with cAMP analogs also leads to an increase in the rate of desensitization of the AChR (23, 24). Taken together, these data suggest that in the intact muscle cell, PKA phosphorylation of the AChR increases the rate of AChR desensitization. The action of PKC-mediated phosphorylation in modulating the AChR has also been examined pharmacologically in cultured muscle cells. Treatment with phorbol esters results in phosphorylation of the AChR on the S subunit in chick myotubes (27), and the S and subunits in rat myotubes (K. Miles, P. Greengard, and R. L. Huganir, unpublished results). Furthermore, treatment of chick myotubes with phorbol esters induces a decreased sensitivity to ACh and an increased rate of receptor desensitization (28). These effects suggest that activation of PKC results in phosphorylation of the AChR and modulation of receptor desensitization. The extracellular signals responsible for activation of the various kinase-mediated pathways have also been investigated. Although skeletal muscle cells are not innervated by the sympathetic nervous system, they express /3-adrenergic receptors which mediate their response to circulating catecholamines. Incubation of BC3H1 myotubes with isoproterenol results in alterations in the phosphorylation state of the a, and S subunits of the AChR, presumably through the activation of PKA (20). Although epinephrine alters muscle cell physiology, the relevance of isoproterenolinduced phosphorylation of the AChR has not been clarified. Another extracellular signal that may stimulate PKA-mediated phosphorylation of the AChR is calcitonin gene-related peptide (CGRP), a neuropeptide that has been identified in presynaptic vesicles of the neuromuscular junction (29). Treatment of cultured rat myotubes with CGRP induces
The FASEB journal
/3,
SWOPE EF AL.
cAMP accumulation and phosphorylation of the AChR on the a and S subunits (30). In addition, CGRP induces an enhancement in the rapid phase of AChR desensitization in cultured mouse muscle cells (24) with a time course similar to that of the CGRP-induced phosphorylation of the AChR. These data suggest that CGRP released from the presynaptic terminal results in PKA phosphorylation of the AChR and modulation of the desensitization rate of the receptor. Several studies during the past few years have identified ACh as a potential extracellular signal that induces PKCmediated phosphorylation and modulation of the AChR.
Using
the cell-attached
patch
configuration
with
ACh
in the recording pipette, ACh applied to the bath decreases the probability of channel opening under the patch. The effect of the bath-applied agonist is blocked by curare (31). Thus, activation of an intracellular second-messenger system by AChR apparently is involved in the regulation of the channel properties of the AChR. Activation of nicotinic cholinergic receptors induces phosphorylation of the AChR S subunit of cultured chick myotubes and the and S subunits of cultured rat myotubes (27; K. Miles, P. Greengard, and R. L. Huganir, unpublished results.) In addition, activation of PKC with phorbol esters mimics the effect of carbamylcholine-stimulated AChR phosphorylation in chick and rat myotubes (27; K. Miles, P. Greengard, and R. L. Huganir, unpublished results). The ACh-induced phosphorylation in rat myotubes is dependent on extracellular calcium and is abolished by AChR channel blockers, suggesting that calcium influx through the AChR is crucial for the activation of PKC (K. Miles, P. Greengard, and R. L. Huganir, unpublished results). Thus, the AChR is a substrate for PKC in intact muscle cells, and PKC appears to be activated by calcium permeation through the AChR in response to agonist. This model presents a novel pathway for autoregulation of the AChR. In contrast to PKC- and PKA-mediated pathways, the extracellular factors involved in regulating tyrosine phosphorylation of the AChR have been elusive. Endogenous tyrosine phosphorylation of the AChR is high in Torpedo electric organ (11, 18) but barely detectable in cultured rat myotubes (16) or BC3H1 cells (20). These observations led to the proposal that tyrosine phosphorylation of the AChR is dependent on innervation. In fact, the AChR at the neuromuscular junction of innervated rat diaphragm is highly phosphorylated on tyrosine residues (see Fig. 2). In addition, tyrosine phosphorylation occurs subsequent to innervation of the muscle during development and is abolished by denervation (32). These findings establish a role for the neuron in regulating tyrosine phosphorylation of the AChR. The extracellular signals released from the nerve that regulate the protein tyrosine kinase have been investigated recently. Agrin, a protein originally isolated from the electric organ of Torpedo, is present in motor neurons and accumulates in the synaptic cleft (33). Agrin is thought to mediate the nerveinduced aggregation of the AChR beneath the nerve terminal. Treatment of cultured chick myotubes with agrin stimulates tyrosine phosphosylation of AChR on the j3 subunit and is associated with AChR aggregation (34). These results suggest that tyrosine phosphorylation of the AChR may play an important role in agrin-induced AChR aggregation. Another extracellular signal that potentially regulates tyrosine phosphorylation of the AChR is basic fibroblast growth factor (bFGF). The bFGF receptor is a known protein tyrosine kinase (35). Treatment of Xenopus myoblast with bFGF bound to latex beads induces AChR cluster formation at the beadmyoblast contact (36). This bFGF-induced clustering is inhibited by an agent that blocks protein tyrosine kinase activity. RECEPTORPHOSPHORYLATION
Figure 2. Visualization of tyrosine phosphorylation of the nicotinic acetyicholine receptor at the rat neuromuscular junction using confocal scanning microscopy. Rat skeletal muscles were immunofluorescently labeled with antibodies to the AChR (red) and to phosphotyrosine (blue). The nicotinic receptor and phosphotyrosine directly colocalize; however, the two fluorescent signals have been artifically separated to allow side-by-side comparison. Photograph contributed by Dr. Zhican Qu.
Thus, tyrosine phosphorylation of the AChR, or some other substrate, appears to mediate bFGF-induced AChR clustering. The mechanism by which agrin and bFGF-induced tyrosine phosphorylation regulate AChR aggregation is not clear. It is possible that tyrosine phosphorylation of the AChR may alter its interaction with the cytoskeleton in such a way as to cause AChR immobilization. Thus, AChR aggregates would accumulate at synaptic sites as a result of the local activation of tyrosine phosphorylation by synaptic agrin. Protein phosphorylation of the AChR has also been proposed to be involved in the regulation of other properties of the AChR. For example, the phosphorylation state of the receptor correlates with both AChR assembly and cellsurface cluster dispersal (37, 27). In cultured chick myoblasts, the S subunit is more highly phosphorylated in the unassembled state compared with the assembled AChR (37). These data suggest that dephosphorylation of the S subunit may be involved in AChR assembly. Treatment of chick muscle cells with phorbol ester or nicotinic agonist stimulates S subunit phosphorylation and decreases the number of AChR
2517
clusters per myotube. However, a concomitant decrease in abungarotoxin binding is not observed, suggesting that cluster dispersal results from lateral migration of the AChRs in the membrane (27). By comparison, activation of PKA with agents that elevate intracellular cAMP levels results in an increase in the number of cell-surface AChRs due to an increased stability and assembly of AChR subunits (38, 39). Thus, although modulation of the rate of desensitization has been most thoroughly studied, additional effects of AChR phosphorylation have recently been reported, including alterations in AChR aggregation and assembly.
NEURONAL REC EPTORS
NICOTINIC
ACETYLCHOLINE
AChRs are also expressed in the central nervous system and autonomic ganglia. Biochemical studies demonstrate that the nAChR from rat and chick brain are 300-kDa complexes of two types of subunits, a and /3 (40, 41). Molecular cloning and sequencing have identified four a (as-as) and three f3 subunits that are highly homologous to the subunits of the muscle AChR (see ref 42 and the references within). No or S subunit homologs have been identified to date. The neuronal AChR is a pentameric complex of these subunits (43) and most likely contains two a subunits and three /3 subunits. Each subunit, like those of the muscle AChR, exhibits four highly conserved putative transmembrane domains and an intracellular loop between M3 and M4 (42). (/32-/344)
Examination of the nAChR intracellular loop reveals putative consensus phosphorylation sites for PKA on the a4 subunit, for PKC on the /3 and /34 subunits, and for protein tyrosine kinases on the a4 subunit (see Table 1). Modulation of nAChRs by phosphorylation has been investigated electrophysiologically and, more recently, biochemically. Activation of PKA or PKC results in functional modulation of the nAChR. In cultured embryonic chicken sympathetic ganglion neurons, phorbol esters enhance the rate of nAChR desensitization without affecting the peak response, suggesting that PKC regulates desensitization of the nAChR (44). In contrast, upon treatment of cultured chick ciliary ganglion neurons with cAMP analogs, the peak whole cell current response to ACh increases two- to threefold. This PKA-dependent mechanism appears to involve an increase in the number of functional nAChRs (45) and may be due to direct phosphorylation of the receptor (46). In support of this model, the AChR of chick ciliary ganglion neurons is phosphorylated on the a3 subunit, and phosphorylation is increased threefold by treatment with cAMP analogs. In addition, the cAMP-induced increase in phosphorylation parallels, in both time course and magnitude, the cAMPinduced increase in peak current response (46). Thus, activation of either PKA or PKC modulates the neuronal AChR properties, although no role for tyrosine phosphorylation in modulating the neuronal AChR has been reported. The identity of extracellular signals involved in the regulation of phosphorylation of nAChRs has also been investigated. The neuropeptide substance P was originally identified as a putative extracellular signal because it enhances agonistinduced nAChR desensitization in PC12 cells (47), primary cultures of bovine chromaflin cells (48), and cultures of sympathetic and parasympathetic neurons (49). nAChRs monitored by cell-attached patches are modulated by bath-applied peptide, suggesting that substance P acts via an intracellular second messenger system (50). The receptors for the substance P family of peptides appear to be coupled to phosphatidylinositol hydrolysis (51), and phorbol esters mimic the
2518
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May 1992
modulatory effect of substance P on agonist-induced desensitization of the nAChR. These results suggest that PKC phosphorylation of the AChR may mediate the effect of substance P on nAChR desensitization (44). However, the action of substance P to increase the rate of nAChR desensitization is not mediated by a classical substance P receptor, as demonstrated by the concentration-dependence and substance-P analog specificity (50). Whether the effects of substance P on the nAChR are mediated by PKC or some other intracellular second-messenger system remains to be clarified. A second putative extracellular signal involved in the modulation of a neuronal AChR via phosphorylation is the neuropeptide vasoactive intestinal peptide (VIP). Treatment of chick ciliary ganglion neurons with VIP increases the AChinduced conductance by 40%, increases intracellular cAMP levels as measured by cAMP imaging techniques, and mimics the effect of intracellularly applied cAMP on the electrophysiological response to ACh (52). Thus, substance P and VIP may act as extracellular first messengers that modulate neuronal AChR function. 7-AMINOBUTYRIC
AC ID (GABAA)
RECEPTORS
GABAA receptors are the receptors for the major inhibitory neurotransmitter in the central nervous system. The structure of the subunits that comprise the GABAA receptor have recently been established by molecular cloning techniques (53). It has become apparent from their structural homology to the muscle and neuronal AChR subunits that these receptors belong to a gene superfamily of ligand-gated ion channels (54). Such structural similarities suggest that the GABAA receptors may also be regulated by protein phosphorylation. Biochemical studies suggest that the purified GABAA receptor from bovine cerebral cortex consists of two types of subunits, a and 3 (55). However, molecular cloning has revealed a multiplicity of GABAA receptor subunits that can be divided into four classes based on sequence homology: a, 3, , and S. To date, six a, three /3,three 7, and one S subunit have been identified (53). GABAA receptors are most likely pentameric complexes of these subunits. It is still unclear which of these subunits comprise GABAA receptors in vivo, although expression of a1, /32, and 72 subunits in heterologous expression systems yields a receptor with many of the properties of the major GABAA receptor subtype present in brain (56). These subunits are predicted to have four transmembrane a-helices (Ml-M4) and a transmembrane topology similar to that of the AChR (see Fig. 1). The GABAA receptor subunits have a large intracellular loop between proposed transmembrane domains M3 and M4 which shows the most sequence divergence among the GABAA receptor subunits. Examination of this intracellular loop region reveals many consensus phosphorylation sites for PKA, PKC, and protein tyrosine kinases, with the number and position of phosphorylation sites varying among the different subunits (Table 1). All /3 subunit cDNAs isolated to date contain a conserved consensus PKA phosphorylation site, and all subunits have a conserved consensus protein tyrosine kinase phosphorylation site. In addition, recent studies have revealed that two differentially spliced forms of the 72 subunit exist in bovine and mouse brain (see Fig. 3 and Table 1). These forms differ by an insertion of eight amino acids in the major intracellular loop that includes a consensus phosphorylation site for PKC (57, 58). This suggests that alternative splicing of the 72 subunit provides a novel mechanism for conveying PKC sensitivity to the GABAA receptor and that receptors containing different forms of the 72
subunit
The FASEBJournal
may be differentially
regulated
by PKC. SWOPE ET AL.
A
2
V C
V C
1=
-I+---1.3kb
3.4kb 24
bp
B p
$ LLRMFSFI(
II1
MYFVSNRKPSKDKDKKKKNPAPTIDIRPRSAT)QMNNATMLOE
Figure 3. Two forms of the GABAA receptor 72 subunit generated by alternative splicing. A) Structure of the 72 subunit gene in the region of the 24-base alternatively spliced exon. B) The amino acid sequence of the M3-M4 cytoplasmic loop of the mouse 72 subunit in the region of the insertion of the alternatively spliced exon. The serine that is a potential PKC phosphorylation Site IS indicated.
Experiments studying the phosphorylation of purified GABAA receptor preparations in vitro have revealed that the GABAA receptor is a substrate for a variety of protein kinases. An unidentified serine protein kinase that is associated with purified preparations of the GABAA receptor has been reported to phosphorylate the a subunit (59). In addition, PKA phosphorylates a /3 subunit of the GABAA receptor (60). Purified preparations of GABAA receptors have recently been shown to be heterogeneous, consisting of several proteins in the 51- to 53-kDa range thought to be a subunits, and several 56- to 58-kDa polypeptides, which are believed to be /3 subunits (55). Two distinct /3 subunits are differentially phosphorylated by PKA and PKC. PKA specifically phosphorylates a 58-kDa j3 subunit whereas PKC phosphorylates a 56-kDa /3 subunit (61). These results suggest that two different subtypes of the 3 subunit in the same receptor or distinct receptors can be differentially regulated by PKA and PKC. The effects of CAMP analogs and forskolin on GABAinduced currents and Cl-flux have recently been examined. The results suggest that cAMP-dependent phosphorylation may regulate GABAA receptor function, although the apparent functional effects of phosphorylation are complex. For example, cAMP-dependent phosphorylation appears to regulate the rate of GABAA receptor desensitization and/or the rate of receptor activation in cortical neurons, hippocampal neurons, and synaptoneurosomes (62-65). In contrast, Ticku et al. (66) failed to find an effect of PKA on GABAA receptor desensitization in mouse spinal cord neurons. As with the AChR, these results have been complicated by the fact that the drugs used to regulate protein kinase activity such as forskolin and cAMP analogs appear to have effects that are independent of protein phosphorylation and are most likely due to a direct interaction with the GABAA receptor (67). These results again emphasize that extreme caution must be used when interpreting the effects of drugs presumed to have indirect effects on ion channel function. This is especially true for hydrophobic drugs used to regulate the levels of second messengers and the activity of protein kinase in intact cells. In spite of the direct effects of these
RECEPTORPHOSPHORYLATION
drugs on the GABAA receptor, recent studies have provided compelling evidence that cAMP-dependent phosphorylation inhibits GABAA receptor function (65, 68). Experiments using whole cell patch-clamp techniques on mouse spinal cord neurons have demonstrated that internal perfusion of the cells with the catalytic subunit of PKA reduces the amplitude of the GABA-evoked current by 60% (65). The effect of the catalytic subunit was also examined in isolated outsideout patches (65). When kinase is included in the pipette, the open channel probability drops dramatically, with the overall GABAA current decreasing by 97%. No significant effect of the kinase on single-channel open time or conductance is observed. Moreover, in recent studies measuring GABA-induced Cl flux in brain microsacs, the introduction of PKA into the microsacs decreases the GABA-induced Cl- flux (68). In addition to the apparent inhibitory effects of cAMPdependent phosphorylation of the GABAA receptor, GABA responses in cerebellar Purkinje cells are potentiated by norepinephrine, an effect that is apparently mediated by cAMP (69). This result is supported by recent findings that GABAinduced currents in dissociated Purkinje cells are potentiated after intracellular perfusion with the catalytic subunit of PKA (H. H. Yeh andJ. E. Cleun, personal communication). The effect of PKC activation on GABAA receptor function has been examined in oocytes expressing ion channels from chick forebrain or rat brain total mRNA (70, 71). PKC activation by phorbol esters and diacylglycerol analogs decreases the amplitude of the GABAA receptor current. These results suggest that PKC phosphorylation of the GABAA receptor may inhibit function in a manner similar to that seen with PKA phosphorylation. However, not all GABAA receptors are regulated by PKC (66, 62). These results are consistent with the known differential splicing of the ‘12 subunit, suggesting that subtypes of the GABAA receptor containing the different 72 subunits may be differentially regulated by PKC. Protein phosphorylation of GABAA receptors has also been suggested to play a role in maintaining GABAA receptor function. When neurons in culture are perfused with minimal intracellular medium, the GABAA conductance progressively decreases with time (72, 73). This rundown can be reduced or prevented by inclusion of ATP in the patch pipette, suggesting that protein phosphorylation is required to maintain GABAA receptor function. This protein kinase activity does not seem to be due to PKA or PKC. Further studies demonstrated that this rundown can be reversed by ATP and Mg2 and that the GABA-induced current could be stabilized by buffering the Ca2 concentration to low levels or through the use of W7, a calmodulin inhibitor (74). These results suggest that a calcium/calmodulin-dependent protein phosphatase, possibly calcineurin (protein phosphatase 2B), may be involved in the rundown of GABAA receptor function.
GLYCINE
RECEPTOR
Glycine
is the
nal the
and
major inhibitory neurotransmitter in the spibrainstem. The structure of several subunits of glycine receptor have recently been elucidated by molecular cloning, revealing that it is homologous to the AChR and the GABAA receptors and belongs to the gene superfamily of ligand-gated ion channels. The cDNA clones isolated to date have been divided into two subunit classes, ct and /3: three a subunits and two /3 subunits have been isolated (75). Examination of the sequence of the major intracellular loop regions of these subunits reveals a number of consensus phosphorylation sites (Table 1), suggesting that cord
2519
the glycine receptor may be regulated by protein phosphorylat ion as well. Biochemical analyses of affinity-purified preparations have shown that the glycine receptor consists of a and /3 subunits assembled into a pentameric complex that contains the ion channel of the receptor (76). Using such receptor preparations, Ruiz-Gomez et al. (77) have demonstrated that the glycine receptor is a substrate for PKC in vitro. PKC specifically phosphorylates Ser-392 of the glycine receptor a subunit (Table 1) (77). The functional effects of this phosphorylation have not yet been determined. Recent studies of spinal trigeminal neurons have provided strong evidence that cAMP-dependent phosphorylation of the glycine receptor modulates its function. Treatment of these neurons with cAMP analogs potentiates the glycineinduced Cl currents (78). Moreover, when the catalytic subunit of PKA is intracellularly perfused, the glycineinduced current is potentiated, an effect due to an increase in the probability of channel opening.
GLUTAMATE
RECEPTORS
The major excitatory neurotransmitter in the CNS is glutamate, and the receptors that mediate transmission across excitatory synapses have been extensively studied pharmacologically, electrophysiologically, and more recently by molecular cloning and expression techniques. The glutamate receptors that function as ligand-gated ion channels can be divided into two main classes, N-methyl-D-aspartate (NMDA) and AMPA/kainate receptors, based on pharmacological and electrophysiological data (79). Recent studies have suggested that these receptors are functionally modulated (80). Of particular interest is the role such modulation is thought to play in synaptic plasticity. In long-term potentiation (LTP), which is a model for learning and memory in the hippocampus, a persistent enhancement of synaptic transmission, lasting from hour to weeks, is induced by the brief repetitive stimulation of monosynaptic excitatory pathways (81). Although much of the potentiation during LTP is thought to be due to a presynaptic mechanism (82, 83), the enhancement appears to be due in part to an increased sensitivity of AMPA/kainate receptors to glutamate (84, 85). The induction of LTP appears to depend on the activity of PKC and calcium/calmodulin-dependent protein kinase II in the postsynaptic neuron (81). Conceivably, the direct phosphorylation of the glutamate receptors could account for their increased sensitivity. In fact, recent electrophysiological studies at the single channel level using rat hippocampal neurons have shown that PKA increases the frequency and duration of kainate-gated channel openings, strongly supporting a direct role for phosphorylation in channel modulation (86). Studies of another system, the white perch retina, have also suggested a role for phosphorylation in altering the channel properties of AMPA/kainate receptors. In cone retinal horizontal cells of these fish, dopamine, which stimulates cAMP production, and cAMP analogs both enhance the ionic conductance activated by kainate, as measured by whole cell voltage-clamp techniques (87). Analysis at the single-channel level demonstrates that the dopaminergic enhancement is due to an increase in the frequencies of kainate-induced channel openings and channel open time (88). Furthermore, perfusion of these cells with the catalytic subunit of PKA also enhances kainate-evoked currents, pointing to a role for direct PKA phosphorylation in these changes (89). 2520
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Another example of synaptic plasticity is long-term depression in the cerebellum (90). When the two excitatory inputs to the Purkinje cells, the climbing fibers and parallel fibers, are conjunctively activated, parallel fiber-Purkinje cell neurotransmission is depressed. At the molecular level, this depression appears to result from the desensitization of AMPA receptors in the postsynaptic Purkinje cells (91, 92). Application of phorbol esters mimics this effect (93). These data suggest that activation of PKC, through calcium influx in response to climbing fiber stimulation and/or intracellular calcium mobilization and diacylglycerol formation in response to metabotropic glutamate receptor stimulation (92), may result in the phosphorylation and subsequent desensitization of AMPA receptors in the Purkinje cells. Physiological studies have recently indicated that protein phosphorylation. regulates the function of NMDA receptors as well (94). Intracellular perfusion of cultured murine hippocampal neurons causes a progressive washout of NMDAbut not kainate-induced current. However, the inclusion of an ATP-regeneration system in the perfusion buffer prevents this washout, whereras GTP or nonhydrolyzable analogs of ATP have no effect. Moreover, this washout can be partially reversed with ATP (94). These results are analogous to those seen with GABA-induced currents and suggest that phosphorylation is required for maintenance of the functional state of the NMDA-gated channel. Although most of these studies used primarily electrophysiological techniques, the recent cloning and functional expression of AMPA/kainate glutamate receptors from the rat brain (79) will allow a more detailed molecular analysis of phosphorylation. Like other ligand-gated ion channels, the glutamate receptors have four putative transmembrane domains (M1-M4), with a large intracellular loop between M3 and M4. However, unlike the AChR, the GABAA receptor, and glycine receptor, the large intracellular loop is the most conserved region among the subtypes (95). This loop contains consensus sites for many protein kinases, including calcium/calmodulin-dependent protein kinase II, PKC, PKA, protein tyrosine kinases, and casein kinases. In particular, the G1uR6 intracellular loop contains a strong consensus site for PKA phosphorylation (Table 1) (96). Whether these sites are actually phosphorylated in vivo as well as any effects that alternative splicing, subunit composition, or ligand binding may have on receptor phosphorylation remain to be addressed. Recent in vitro studies of a purified chick cerebellar kainate-binding protein have shown that PKA phosphorylation is negatively regulated by ligand binding (97). From analysis of the cDNA sequence (98), this protein appears to have many characteristics of a ligand-gated ion channel subunit, including four transmembrane domains and consensus sites for PKA, PKC, and protein tyrosine kinases in the predicted intracellular loop. A similar protein from frog has consensus sites for PKA and protein tyrosine kinases (99). Both proteins share some sequence identity with the rat glutamate receptor cDNAs. However, whether these proteins are subunits of functional channels has not yet been convincingly demonstrated.
SUMMARY
AND
CONCLUSIONS
Most ligand-gated ion channels characterized thus far have been shown to be regulated by protein phosphorylation or to contain consensus phosphorylation sites for protein kinases. Phosphorylation of these receptors occurs on the major intracellular loop between the third and fourth transmembrane domain of the receptor subunits, and many subunits contain
The FASEBJournal
SWOPE ET AL.
multiple phosphorylation sites for a single protein kinase or for several distinct protein kinases. Many ligand-gated ion channels appear to be phosphorylated by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) as well as by protein tyrosine kinases (PTK). These kinase systems are regulated by a variety of extracellular signals released from the presynaptic neuron (see Fig. 4). PKA phosphorylation of the receptors is regulated by first messengers that increase the intracellular levels of cAMP, such as the neuropeptides CGRP and VIP in the case of the muscle AChR and neuronal AChR, respectively (see Fig. 4). In addition, the neurotransmitters dopamine and norepinephrine regulate PKA phosphorylation of glutamate and GABAA receptors, respectively. PKC phosphorylation of the receptors is regulated by first messengers which increase the turnover of phosphatidylinositol and/or increase the intracellular levels of calcium. Substance P and glutamate working through G proteinlinked receptors can stimulate PKC phosphorylation of neuronal AChRs and non-NMDA glutamate receptors, respectively. In addition, activation of the AChR ion channel by acetylcholine allows calcium to enter the cell and directly activate PKC, resulting in the phosphorylation of the AChR (see Fig. 4). This type of mechanism is similar to that proposed for the NMDA receptor, where activation of the NMDA receptor by glutamate allows calcium entry, directly activating
PKC and/or
calcium/calmodulin-dependent
protein
kinase
II (CAM-K) during LTP. However, the pathways involved in regulating tyrosine phosphorylation of the receptors are less clear. In the case of the muscle AChR, the extracellular matrix protein, agrin, and possibly bFGF regulate tyrosine phosphorylation. As agrin and bFGF are highly expressed in the central nervous system, and other ligand-gated ion chan-
Dopamine CGRP VIP
Substance P Glutamate (Quis)
Agnn
Acetylcholine
bFGF
‘Glutamate (NMDA)
nels contain
consensus sites for tyrosine phosphorylation, a may also exist for the regulation of the other ligand-gated ion channels. In spite of the fact that most of the phosphorylation sites on ligand-gated ion channels are contained within the major intracellular loop between M3 and M4 transmembrane domains, the functional effects of phosphorylation are diverse. Phosphorylation in this region appears to regulate desensitization, open-channel probability, and aggregation of receptors as well as the assembly of receptor subunits (see Fig. 4). The molecular mechanisms responsible for the diversity of functional effects are not clear. It is possible that the intracellular loops of various subunits play distinct functional roles, and consequently phosphorylation of different subunits has distinct functional effects. In addition, the intracellular loop in most of the receptor subunits is relatively large and may consist of several distinct functional domains that could be separately regulated by phosphorylation. For example, in the case of the neuronal AChR and the GABAA receptor, phosphorylation of the receptors by different protein kinases has been reported to have opposing effects on the properties of the ion channel. It is possible that phosphorylation of these receptors by the different protein kinases occurs on distinct subunits, thereby producing different changes on the channel properties, or alternatively, that the two kinases could phosphorylate different regions within the intracellular loop of the same subunit. Moreover, in the case of the GABAA receptor, phosphorylation of the receptors by the same protein kinase has been reported to have opposite effects in different cell types. One hypothesis to account for these results is that the two GABAA receptors contain different subunit subtypes that have PKA phosphorylation sites in distinct regions of their intracellular loop. In the future, questions such as these will be clarified by studying the phosphorylation and regulation of receptors containing identified subunits in heterologous expression systems using site-specific mutagenesis techniques. Such complexity in the regulation of ligand-gated ion channels by phosphorylation suggests that by altering the expression of receptor subunit subtypes and various protein kinases, neurons can finely tune their synapses. Changes in receptor desensitization or open-channel probability would have profound effects on the efficacy of synaptic transmission. In addition, regulation of receptor density at the postsynaptic membrane through an alteration in the aggregation of receptors or the local assembly of the subunits could modify the postsynaptic response. Finally, the ability of other signaling pathways at the synapse to modulate the phosphorylation of the receptors allows these properties to be dynamically regulated, resulting in short- or long-term changes in synaptic transmission. similar
pathway
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Figure 4. Modulation of ligand-gated ion channels at an idealized composite synapse by protein phosphorylation systems. Extracellutar signals released from the presynaptic neuron interact with specific receptors on the postsynaptic cell and indirectly activate various protein kinase systems, resulting in the phosphorylation and functional modulation of the ligand-gated ion channels (see Summary and Conclusion for discussion).
RECEPTOR PHOSPHORYLATION
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