Depolarization of Rat Brain Synaptosomes Increases Phosphorylation of Voltage-sensitive Sodium Channels*

(Received for publication, January 2, 1997, and in revised form, May 6, 1997)

Tamara Kondratyuk Dagger and Sandra Rossie §

From the Departments of Dagger  Veterinary Pathobiology and § Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Depolarization of rat brain synaptosomes causes an increase in phosphorylation of serine residues 573, 610, 623, and 687 on voltage-sensitive sodium channels. Although these sites have been shown to be phosphorylated by cAMP-dependent protein kinase in vitro and in situ, the depolarization-induced increase in their state of phosphorylation is not due to increased cAMP-dependent protein kinase activity, but requires calcium influx and protein kinase C. Since phosphorylation at this cluster of sites inhibits sodium current and would decrease neuronal excitability, this may be an important negative feedback mechanism whereby calcium influx during prolonged or repetitive depolarization can attenuate neuronal excitability and prevent further calcium accumulation. Phosphorylation of purified channels by protein kinase C decreases dephosphorylation of cAMP-dependent phosphorylation sites by purified calcineurin or protein phosphatase 2A. This suggests that one mechanism by which protein kinase C may increase phosphorylation of cAMP-dependent phosphorylation sites in sodium channels is to inhibit their dephosphorylation. This represents an important new mechanism for convergent regulation of an ion channel by two distinct signal transduction pathways.


INTRODUCTION

Sodium influx through voltage-sensitive sodium channels is responsible for the initiation and propagation of action potentials in most neurons, and ultimately promotes the opening of voltage-sensitive calcium channels at the synapse, leading to calcium influx and exocytosis. Because of their central role in these processes, the response properties of sodium channels can control neuronal excitation and secretion. Phosphorylation of brain sodium channels by PKA1 and PKC dramatically alters their electrophysiological responses. Cyclic AMP-dependent phosphorylation decreases sodium current in response to membrane depolarization (1, 2) due to a decrease in the probability of channel opening (1). Phosphorylation by PKC slows channel inactivation (3), decreases sodium current (3-5), and is required for the electrophysiological effect of cAMP-dependent phosphorylation to be observed (6). Two electrophysiological studies have demonstrated that neurotransmitters modulate sodium channel function via second messenger-induced phosphorylation. Stimulation of D1 dopamine receptors in dissociated rat nigrostriatal neurons reduces sodium current in a cAMP-dependent manner (7). Stimulation of muscarinic receptors in cultured hippocampal neurons, via activation of PKC, also reduces sodium current (8).

The alpha  subunit of brain sodium channels is phosphorylated by PKA on multiple serine residues clustered in a single intracellular loop between homologous domains I and II of the channel protein (9-11). Replacement of this loop with the corresponding loop from a skeletal muscle sodium channel lacking these phosphorylation sites abrogates the cAMP-mediated inhibition of channels expressed in Xenopus oocytes (12). Thus, this intracellular segment contains all phosphorylation sites necessary for cAMP-dependent inhibition of brain sodium channels. Li and co-workers (6) found that the effect of 8-bromo-cAMP on sodium current could not be observed in Chinese hamster ovary cells expressing sodium channels without prior activation of PKC. Ser-1506, located in the intracellular loop between homologous domains III and IV, was the critical phosphorylation site for PKC in causing this effect (6). Thus, sodium channels are a target for convergent regulation by two distinct second messenger-regulated pathways causing phosphorylation in two different regions of the channel protein. Although PKC-mediated channel phosphorylation appears to be a prerequisite for cAMP-dependent reduction in sodium current, it is not known whether PKC phosphorylation alters the sodium channel's response to cAMP-dependent phosphorylation or whether it alters the channel's susceptibility to phosphorylation or dephosphorylation at cAMP-dependent phosphorylation sites.

In the present study, we observed that depolarization of rat brain synaptosomes caused a calcium-dependent increase in phosphorylation of cAMP-dependent phosphorylation sites on sodium channels. This response was not due to increased PKA activity, but required activation of PKC. These results suggest that accumulation of calcium and activation of PKC in nerve terminals may lead to increased phosphorylation and consequent decreased responsiveness of sodium channels. We also found that purified reconstituted sodium channels phosphorylated by both PKC and PKA were poorly dephosphorylated by purified calcineurin or PP2A, in contrast to sodium channels phosphorylated by PKA alone. This observation suggests one potential mechanism by which PKC activation during synaptosome depolarization could lead to an increase in phosphorylation of cAMP-dependent phosphorylation sites on sodium channels. The ability of one phosphorylation site on a given substrate to control dephosphorylation of a separate site represents a potentially important molecular mechanism for conditional or convergent regulation of the responses of ion channels and other phosphoproteins by distinct signal transduction pathways.


EXPERIMENTAL PROCEDURES

Materials, Purified Proteins, and Antibodies

Sodium channels were purified through the wheat germ-agglutinin chromatography step according to Hartshorne and Catterall (13). Heterotrimeric PP2A containing a mixture of PP2A1 and PP2A0 was purified from rat brain as described previously (14). The catalytic subunit of PKA was purified from bovine heart (15). Monoclonal antibody 1G11 generated toward purified rat brain sodium channel was kindly provided by Dr. William Catterall (University of Washington). Recombinant mouse PKCalpha (16), expressed in Sf9 cells (17) and partially purified from Sf9 cell lysates by anion exchange chromatography was kindly provided by Dr. Curt Ashendel (Purdue University). Other biochemicals and reagents were purchased from the following sources: purified calcineurin, Upstate Biotechnology Inc.; N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin, Worthington; phosphocellulose paper, Whatman; [gamma -32P]ATP, NEN Life Science Products; protein kinase inhibitor, which specifically inhibits PKA, Sigma; forskolin, PMA, MPMA, BIM, (RP)-cAMPS, okadaic acid, lipids, and cyclosporin A, Calbiochem.

Synaptosome Preparation and Treatments

All procedures were performed at 0-4 °C unless otherwise noted. Synaptosomes were freshly prepared from adult male rat brains as described previously (18) and resuspended in (20 mM HEPES, pH 7.4, 10 mM glucose, 132 mM NaCl, 4.8 mM KCl, 2.4 mM MgSO4, 0.1 mM EGTA, 1.2 mM CaCl2) to a final protein concentration of approximately 1.5 mg/ml. Samples (50-150 µg protein) were preincubated for 5 min at 37 °C, then treated with drugs or appropriate vehicle for various times before depolarization. To depolarize, synaptosomes were diluted 2-fold with prewarmed (20 mM HEPES, pH 7.4, 10 mM glucose, 22 mM NaCl, 115 mM KCl, 2.4 mM MgSO4, 0.1 mM EGTA, 1.2 mM CaCl2) for a final concentration of 60 mM potassium and incubated for 1 min at 37 °C. When examining the requirement for calcium, CaCl2 was omitted from each buffer.

Isolation and Back Phosphorylation of Sodium Channels

After treatment samples were solubilized, and sodium channels were immunoprecipitated, back-phosphorylated with the catalytic subunit of PKA and [gamma -32P]ATP, then subjected to SDS-PAGE and autoradiography as described previously (18). Gel slices containing sodium channel alpha  subunits were excised and dissolved, and their 32P content was quantified by liquid scintillation spectrometry.

Two-dimensional Phosphopeptide Maps

Two-dimensional tryptic phosphopeptide maps were generated from SDS-polyacrylamide gel electrophoresis gel bands containing 32P-labeled sodium channel alpha  subunits as described previously (19, 20) or, in some cases, by digesting after electrophoretic transfer to nitrocellulose as described by Luo and colleagues (21).

Measurement of PKA Activity

Protein kinase A activity was measured according to the protocol of Roskoski (22) with some modifications. Briefly, after treatment synaptosomes were lysed with an equal volume of (100 mM Tris, pH 7.4, 0.2 mM EDTA, 0.2 mM EGTA, 9% Triton X-100). An aliquot of the lysate was mixed with assay buffer in a final volume of 30 µl containing 50 mM Tris, pH 7.4, 4 mM magnesium acetate, 50 mM beta -glycerophosphate, 0.1 mM 3-isobutyl-1-methylxanthine, 2 mM EGTA, 1.5% Triton X-100, 1 mg/ml bovine serum albumin, 200 µM [gamma -32P]ATP (2000 cpm/pmol), 1 mg/ml histone 2A, and incubated for 5 min at 30 °C. An aliquot of each sample was spotted onto phosphocellulose paper, washed in 75 mM H3PO4, and air-dried, and 32P was quantified by liquid scintillation spectrometry.

Reconstitution and Phosphorylation of Sodium Channels

Purified rat brain sodium channels were reconstituted into phospholipid vesicles and phosphorylated with the purified catalytic subunit of PKA, with PKC, or with both kinases as described by Murphy and Catterall (20) with minor modifications. To label cAMP-dependent phosphorylation sites, reconstituted channels (1-4 pmol) were incubated at 30 °C for 2 h with the catalytic subunit of PKA (0.5 µg/ml) in 295 mM sucrose, 50 mM Tris-HCl, pH 7.4, 50 mM Na2SO4, 10 mM MgCl2, 1 mM EGTA, and 160 µM [gamma -32P]ATP (1-2 Ci/mmol). To label PKC phosphorylation sites, reconstituted channels were treated at 30 °C for 2 h with recombinant PKC (1 µg/ml) in 295 mM sucrose, 50 mM Tris-HCl, pH 7.4, 50 mM Na2SO4, 10 mM MgCl2, 1.5 mM CaCl2, 60 µM phosphatidylserine, 1.5 µM diolein, and 1 µg of protein kinase inhibitor, to inhibit endogenous PKA activity present in wheat germ-agglutinin purified channel preparations (20). To phosphorylate with both kinases, reconstituted channels were treated first with PKC without the addition of protein kinase inhibitor for 2 h, then the catalytic subunit of PKA and EGTA (to a final concentration of 10 mM) were added, and the incubation was continued for an additional 2 h. Phosphorylated reconstituted channels were passed through Sephadex G-50 equilibrated in 221 mM sucrose, 50 mM Tris-HCl, pH 7.4, 75 mM Na2SO4, 0.15 mM MgCl2, then used in phosphatase assays. Phosphopeptide maps of phosphorylated reconstituted channels indicated that the patterns of phosphorylation by PKC and PKA were similar to those previously reported (19, 20) (data not shown). The average stoichiometry of channel phosphorylation (expressed as moles of phosphate/mol of channel) was 3, 2, and 4.2 for phosphorylation by PKA, PKC, or both kinases, respectively.

Phosphatase Assay

Phosphorylated reconstituted sodium channels were mixed in a 30-µl final volume containing either PP2A (50 ng) in the presence of (0.1 mM EDTA, 0.1 mM EGTA, 0.1% beta -mercaptoethanol) or with calcineurin (100 ng) in the presence of (0.75 mM dithiothreitol, 10 mM MgCl2, 0.1 mM CaCl2, 100 nM calmodulin, 500 nM okadaic acid). Okadaic acid was included during treatment with calcineurin to inhibit low levels of okadaic acid-sensitive phosphatase activity present in wheat germ-agglutinin purified sodium channel preparations.2 Samples were incubated at 37 °C for varying amounts of time, then the reaction was terminated with 100 µl of 10% trichloroacetic acid and 10 µl of 10 mg/ml bovine serum albumin. Protein was then sedimented, and acid-soluble [32P]Pi was quantified.


RESULTS

Depolarization of Synaptosomes Increases the Phosphorylation of cAMP-dependent Phosphorylation Sites in Sodium Channels

Back phosphorylation (23) was used to measure changes in phosphorylation of cAMP-dependent phosphorylation sites on sodium channels. In this technique, endogenous phosphate is incorporated into channels in synaptosomes during experimental treatments, then channels are isolated under conditions preventing dephosphorylation and treated with the catalytic subunit of PKA and [gamma -32P]ATP to incorporate radioactive phosphate into sites that were not phosphorylated in situ. Thus, a decrease in back phosphorylation reflects an increase in endogenous phosphorylation. Back phosphorylation of immunopurified sodium channels with the catalytic subunit of PKA specifically labels serine residues 573, 610, 623, and 687 of rat brain type IIA sodium channels (11). Treatments that elevate cAMP in synaptosomes (24) or cultured rat brain cells (19) block subsequent back phosphorylation of these sites, indicating that these sites are phosphorylated by PKA in situ. Serine residues 573, 610, 623, and 687 will be collectively referred to as cAMP-dependent phosphorylation sites in this study. It is not known whether other kinases also phosphorylate these sites in vivo.

Depolarization of synaptosomes by high potassium in the presence of calcium decreased back phosphorylation of channel cAMP-dependent phosphorylation sites, indicating an increase in endogenous phosphorylation (Fig. 1). The magnitude of this response varied from experiment to experiment, but ranged from 25 to 50% in 30 independent experiments. In the absence of calcium no change in phosphorylation occurred, suggesting that the increase in phosphorylation during depolarization requires calcium influx.


Fig. 1. Back phosphorylation of sodium channels following depolarization of synaptosomes in the presence and absence of calcium. Synaptosomes were incubated in buffer containing 4.8 mM K+ (control) or 60 mM K+ (depolarization), in the presence or absence of Ca2+. After treatment, channels were isolated and back phosphorylated. Results are expressed as the percent decrease in back phosphorylation compared with control samples incubated in the presence of Ca2+, and represent the average ± S.E. of three experiments.
[View Larger Version of this Image (15K GIF file)]

The Depolarization-induced Increase in Channel Phosphorylation Is Not Due to an Increased PKA Activity

The role of PKA in this response was examined. Depolarization of synaptosomes did not increase PKA activity (Fig. 2). In addition, the increase in channel phosphorylation caused by depolarization was not blocked by pretreatment with the inhibitory cAMP derivative, (RP)-cAMPS (25) (Fig. 3). In contrast, forskolin increased PKA activity in synaptosomes and decreased back phosphorylation of sodium channels; both of these responses were blocked by (RP)-cAMPS (Figs. 2 and 3). Treatment with (RP)-cAMPS alone had no effect on control kinase activity or channel phosphorylation. These results indicate that the depolarization-induced increase in phosphorylation of cAMP-dependent phosphorylation sites on sodium channels is not caused by increased PKA activity. However, since (RP)-cAMPS may not completely block basal PKA activity (26), basal PKA activity may still be responsible for the accumulation of phosphate in cAMP-dependent phosphorylation sites that occurs during depolarization.


Fig. 2. The effect of forskolin treatment or depolarization on PKA activity in synaptosomes. Synaptosomes were incubated with or without 10 µM (RP)-cAMPS for 10 min, then 10 µM forskolin or vehicle was added and the incubation continued for an additional 15 min, or the incubation was continued for 14 min, then synaptosomes depolarized for 1 min. After treatment, all samples were lysed and PKA activity was measured. Results are the average ± S.E. of three experiments.
[View Larger Version of this Image (15K GIF file)]


Fig. 3. The effect of (RP)-cAMPS on sodium channel phosphorylation during depolarization or treatment with forskolin. Synaptosomes were treated as described in Fig. 2. After treatment, channels were isolated and back phosphorylated. Results are expressed as the percent decrease in back phosphorylation compared with control samples and represent the average ± S.E. of three experiments.
[View Larger Version of this Image (14K GIF file)]

The Response to Depolarization Requires PKC and Calcium

Because depolarization of synaptosomes increases PKC-mediated phosphorylation (27, 28) and sodium channels are a potential substrate for PKC phosphorylation in synaptosomes (29), the involvement of PKC in this response was examined. Treatment of synaptosomes with PMA (100 ng/ml) mimicked the effect of depolarization on back phosphorylation of sodium channels (Fig. 4A). The response to depolarization or PMA treatment was blocked by BIM (1 µM), a specific inhibitor of PKC (30, 31), suggesting that the effect of depolarization on sodium channel phosphorylation is mediated by PKC. The response to PMA, like that to depolarization, was prevented by removal of buffer calcium. Treatment of synaptosomes with BIM alone (Fig. 4A) or with the inactive phorbol ester MPMA (Fig. 4B) had no effect on channel phosphorylation.


Fig. 4. The effect of BIM on sodium channel phosphorylation during depolarization or treatment with PMA. A, synaptosomes were incubated with or without 1 µM BIM for 20 min, then 100 ng/ml PMA or vehicle was added and the incubation continued for an additional 10 min, or the incubation was continued for 9 min, then synaptosomes were depolarized for 1 min. After treatment, channels were isolated and back phosphorylated. Results are expressed as the percent decrease in back phosphorylation compared with control samples and represent the average ± S.E. of three experiments. B, synaptosomes were treated with 100 ng/ml PMA or 200 ng/ml MPMA in the presence or absence of Ca2+. After treatment, channels were isolated and back-phosphorylated. Results are expressed as the percent decrease in back phosphorylation compared with control samples incubated in the presence of Ca2+ and represent the average ± S.D. of two experiments performed in triplicate.
[View Larger Version of this Image (19K GIF file)]

Protein kinase C and PKA both phosphorylate Ser-610 in vitro, but other phosphorylation sites are not shared (20). To determine whether the increase in endogenous phosphorylation at cAMP-dependent phosphorylation sites was due solely to phosphorylation of Ser-610, tryptic phosphopeptide maps of back phosphorylated sodium channel alpha  subunits were prepared and the changes in phosphorylation for each of the four cAMP-dependent phosphorylation sites were quantified. As shown in Fig. 5, depolarization affected all four cAMP-dependent phosphorylation sites to a similar extent. This demonstrates that the depolarization-induced increase in phosphorylation of cAMP-dependent phosphorylation sites is not solely due to the direct phosphorylation of Ser-610 by PKC. However, we cannot rule out the possibility that PKC phosphorylates additional cAMP-dependent phosphorylation sites in vivo, since each of these sites are also potential phosphorylation sites for PKC (32). Circumstances in vivo, such as the presence of a particular subtype of PKC (33) or its strategic localization by A kinase anchoring proteins (34), may permit PKC-mediated phosphorylation of cAMP-dependent phosphorylation sites in addition to Ser-610.


Fig. 5. Analysis of individual cAMP-dependent phosphorylation sites of sodium channels after depolarization of synaptosomes. Sodium channels from control or depolarized synaptosomes were isolated and back phosphorylated, then tryptic phosphopeptide maps were prepared from 32P-labeled sodium channel alpha  subunits. The 32P content of phosphopeptides corresponding to each cAMP-dependent phosphorylation site was quantified. Results are expressed as the percent decrease in back phosphorylation compared with control samples and represent the average ± S.E. of three experiments.
[View Larger Version of this Image (20K GIF file)]

Phosphorylation of Purified Channels by PKC Inhibits Dephosphorylation of cAMP-dependent Phosphorylation Sites by PP2A and Calcineurin

Since the state of phosphorylation of a protein in situ reflects the competing activities of kinases and phosphatases, the effect of phosphatase inhibitors on sodium channel phosphorylation in synaptosomes was evaluated. Previous studies have shown that okadaic acid, which inhibits PP2A and related phosphatases, and cyclosporin A, which can inhibit calcineurin, both increase endogenous phosphorylation of sodium channels in synaptosomes (18). The effect of treatment with each of these agents was not additive with the effect of depolarization (Fig. 6), suggesting that the response to depolarization may be equivalent to blocking dephosphorylation.


Fig. 6. The effect of phosphatase inhibitors and depolarization on sodium channel phosphorylation. Synaptosomes were treated with 300 nM okadaic acid (O.A.) for 10 min or 1 µM cyclosporin A for 30 min, and depolarized (depol) or treated with control buffer during the last minute of incubation. After treatment, channels were isolated and back phosphorylated. Results are expressed as the percent decrease in back phosphorylation compared with control samples and represent the average ± S.E. of three experiments.
[View Larger Version of this Image (14K GIF file)]

A decrease in dephosphorylation may arise from direct inhibition of phosphatases or from a decrease in channel susceptibility to the action of phosphatases. To test this second possibility, the ability of phosphatases to dephosphorylate sodium channels phosphorylated by both PKA and PKC was examined. Purified sodium channels were reconstituted and phosphorylated with each kinase alone, or with both kinases, then used as substrates for dephosphorylation. As in the case of soluble sodium channels (11), reconstituted sodium channels phosphorylated with PKA alone could be dephosphorylated by both calcineurin and PP2A (Fig. 7). In contrast, channels phosphorylated with PKC alone, or with both PKC and PKA, were poorly dephosphorylated by either phosphatase. This suggests that PKC phosphorylation of sodium channels decreases the ability of both calcineurin and PP2A to attack cAMP-dependent phosphorylation sites.


Fig. 7. Dephosphorylation of reconstituted purified sodium channels after phosphorylation with PKA alone, PKC alone, or PKA and PKC. Purified sodium channels were reconstituted and phosphorylated with the catalytic subunit of PKA (bullet ), with PKC (open circle ), or with both kinases (black-down-triangle ), then treated with calcineurin (A) or PP2A (B), and phosphate release was assessed as described under "Experimental Procedures." Results are expressed as moles of Pi released per mol of phosphoserine present in each reaction and represent the average ± S.E. of seven experiments.
[View Larger Version of this Image (16K GIF file)]


DISCUSSION

This study suggests that an important physiologic stimulus, neuronal depolarization, influences the responsiveness of voltage-dependent brain sodium channels by controlling the state of phosphorylation of cAMP-dependent phosphorylation sites. In addition, two novel aspects of the role of PKC in controlling sodium channel phosphorylation are revealed; PKC is required for the effect of synaptosomal depolarization on sodium channel phosphorylation, and the direct phosphorylation of purified sodium channels by PKC reduces the ability of phosphatases to attack cAMP-dependent phosphorylation sites in vitro. Thus, activation of PKC influences the extent of phosphorylation at cAMP-dependent phosphorylation sites on voltage-sensitive sodium channels in brain.

The mechanism by which depolarization increases sodium channel phosphorylation is not known. Our results with purified sodium channels suggest that a PKC-induced decrease in channels' susceptibility to dephosphorylation could play a role in elevating phosphorylation of cAMP-dependent phosphorylation sites on sodium channels in response to depolarization. However, the circumstances in synaptosomes are complex due to the presence of multiple Ser/Thr phosphatases and second messenger-regulated kinases, and other mechanisms may also lead to increased sodium channel phosphorylation. These include the possibility that PKC itself or a kinase activated by PKC directly phosphorylates cAMP-dependent phosphorylation sites on sodium channels in situ, and the possibility that activation of PKC may decrease phosphatase activity in synaptosomes. For example, PKC has been shown to phosphorylate and inhibit calcineurin in vitro (35), and a recent study suggests that activation of PKC may modulate PP2A activity in cardiac myocytes (36). These potential mechanisms remain to be investigated in synaptosomes.

Although it is not clear whether the role of PKC in the depolarization-induced elevation of sodium channel phosphorylation is due to a direct phosphorylation of sodium channels or is indirect, it is unlikely that PKC acts solely by promoting calcium influx or membrane depolarization. Although PKC inhibition during synaptosome depolarization decreases calcium influx and membrane depolarization to a moderate extent (37), treatment of synaptosomes with phorbol ester has little or no effect on calcium levels (38, 39) or membrane depolarization (39) and does not enhance either of these responses to treatment with high potassium (37). In the present study, PMA increased sodium channel phosphorylation in the absence of a depolarizing stimulus, conditions under which calcium elevation is not expected (38, 39). Although the effect of PMA was prevented by incubating synaptosomes in a calcium-free medium, the loss of responsiveness may be explained by a depletion of calcium in synaptosomes during treatment with calcium-free buffer containing EGTA. Others have demonstrated that PKC can phosphorylate different synaptosome substrates depending on the means of activation (40). To determine whether PKC directly phosphorylates sodium channels during depolarization, the phosphorylation of Ser-1506 should be measured. Although phosphorylation of this site in vivo has not yet been examined, it is phosphorylated by PKC but not PKA in vitro (20) and this site plays a key role in the electrophysiological effects of PKC on sodium channels (6).

In addition to its essential role in initiating exocytosis, depolarization-induced calcium influx can modulate the subsequent excitability of the synaptic terminal by activating a host of calcium-dependent enzymes which can alter ion channel function (41, 42). Because phosphorylation at cAMP-dependent phosphorylation sites inhibits brain sodium channels, the enhanced state of channel phosphorylation after depolarization would be expected to inhibit subsequent synaptic excitation, decreasing further calcium influx and secretion. However, a number of studies suggest that activation of PKC enhances neurosecretion (reviewed in Refs. 39 and 43). One possible explanation reconciling these two views may be that rapid or early PKC-mediated phosphorylation events enhance secretion, whereas the increase in channel phosphorylation at cAMP-dependent sites that we observe may require periods of repetitive or sustained depolarization. Early PKC-mediated events may include the phosphorylation of sodium channels at Ser-1506. Phosphorylation at this site slows channel inactivation and would be expected to prolong depolarization and promote calcium influx. Thus, one possible scenario may be that depolarization and calcium influx activates PKC, which phosphorylates a number of target proteins, including sodium channels on Ser-1506, enhancing neurosecretion. During repetitive or prolonged depolarization, the phosphorylation of cAMP-dependent phosphorylation sites is gradually enhanced due to increased phosphorylation, decreased dephosphorylation, or both. This leads to inhibition of sodium channels during subsequent depolarizing stimuli. This may represent a negative feedback mechanism by which neurons are protected from accumulating dangerously high levels of calcium during periods of prolonged depolarization or hyperexcitation.

Several cases have been described in which phosphorylation of one site on a given substrate controls the phosphorylation of another site (44). Although fewer examples of one phosphorylation site controlling the dephosphorylation of a separate phosphorylation site have been reported, several important cases have recently been described. These include an enhanced rate of phosphotyrosine dephosphorylation in the presence of a nearby phosphothreonine residue on mitogen-activated protein kinase by dual-specificity phosphatases (45, 46), the inhibition of calcineurin-mediated dephosphorylation of neuronal DARPP-32 at Thr-34 by casein kinase I-mediated phosphorylation at Ser-137 (47), and the resistance to dephosphorylation of PKCalpha induced by phosphorylation of Thr-638 (48). Such interaction between phosphorylation sites represents an important molecular mechanism for the conditional regulation of a given response. Our results with purified sodium channels suggest that the ability of phosphatases to dephosphorylate cAMP-dependent phosphorylation sites on sodium channels is modulated by PKC phosphorylation of the channel itself. This effect may contribute to the convergent regulation of sodium channel function by PKC and PKA, observed in electrophysiological studies (6), and to the increased phosphorylation of cAMP-dependent phosphorylation sites during synaptosomal depolarization. Since PKC phosphorylation of Ser-1506 is critical for cAMP-dependent inhibition of sodium channel function (6), it will be important to learn if phosphorylation of Ser-1506 is responsible for the effect on dephosphorylation of cAMP-dependent phosphorylation sites observed in vitro.


FOOTNOTES

*   This work was supported by United States Public Health Service Grant NS31221.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This is journal paper no. 15276 from the Purdue University Agricultural Experiment Station.


   To whom correspondence should be addressed: Biochemistry Dept., Purdue University, West Lafayette, Indiana 47907-1153. Tel.: 765-494-3112; Fax: 765-494-7897.
1   The abbreviations used are: PKA, protein kinase A; PKC, protein kinase C; PP2A, protein phosphatase 2A; (RP)-cAMPs, adenosine 3',5'-cyclic monophosphorothiolate-(RP); BIM, bisindolylmaleimide; PMA, phorbol 12-myristate 13-acetate; MPMA, 4-O-methylphorbol 12-myristate 13-acetate.
2   B. Law and S. Rossie, unpublished observation.

ACKNOWLEDGEMENTS

We thank Dr. William Catterall for providing sodium channel antibodies, Dr. Curt Ashendel for providing recombinant PKC, Anna Kempa for purifying sodium channels, Laura Lofland for preparing this manuscript, and Drs. David Armstrong and Stan Rane for critically reading the manuscript.


REFERENCES

  1. Li, M., West, J. W., Lai, Y., Scheuer, T., and Catterall, W. A. (1992) Neuron 8, 1151-1159 [Medline] [Order article via Infotrieve]
  2. Gershon, E., Weigl, L., Lotan, I., Schreibmayer, W., and Dascal, N. (1992) J. Neurosci. 12, 3743-3752 [Abstract]
  3. Numann, R., Catterall, W. A., and Scheuer, T. (1991) Science 254, 115-118 [Medline] [Order article via Infotrieve]
  4. Schreibmayer, W., Dascal, N., Lotan, I., Wallner, M., and Weigl, L. (1991) FEBS Lett. 291, 341-344 [CrossRef][Medline] [Order article via Infotrieve]
  5. Dascal, N., and Lotan, I. (1991) Neuron 6, 165-175 [Medline] [Order article via Infotrieve]
  6. Li, M., West, J. W., Numann, R., Murphy, B. J., Scheuer, T., and Catterall, W. A. (1993) Science 261, 1439-1442 [Medline] [Order article via Infotrieve]
  7. Surmeier, D. J., and Kitai, S. T. (1993) Prog. Brain Res. 99, 309-324 [Medline] [Order article via Infotrieve]
  8. Cantrell, A. R., Ma, J. Y., Scheuer, T., and Catterall, W. A. (1996) Neuron 16, 1019-1026 [CrossRef][Medline] [Order article via Infotrieve]
  9. Rossie, S., Gordon, D., and Catterall, W. A. (1987) J. Biol. Chem. 262, 17530-17535 [Abstract/Free Full Text]
  10. Rossie, S., and Catterall, W. A. (1989) J. Biol. Chem. 264, 14220-14224 [Abstract/Free Full Text]
  11. Murphy, B. J., Rossie, S., De Jongh, K. S., and Catterall, W. A. (1993) J. Biol. Chem. 268, 27355-27362 [Abstract/Free Full Text]
  12. Smith, R. D., and Goldin, A. L. (1996) J. Neurosci. 16, 1965-1974 [Abstract]
  13. Hartshorne, R. P., and Catterall, W. A. (1984) J. Biol. Chem. 259, 1667-1675 [Abstract/Free Full Text]
  14. Law, B., and Rossie, S. (1995) J. Biol. Chem. 270, 12808-12813 [Abstract/Free Full Text]
  15. Kaczmarek, L. K., Jennings, K. R., Strumwasser, F., Nairn, A. C., Walter, U., Wilson, F. D., and Greengard, P. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7487-7491 [Abstract]
  16. Megidish, T., and Mazurek, N. (1989) Nature 342, 807-811 [CrossRef][Medline] [Order article via Infotrieve]
  17. Summers, M. D., and Smith, G. E. (1987) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures., Texas Agricultural Experiment Station Bulletin No. 1555
  18. Chen, T., Law, B., Kondratyuk, T., and Rossie, S. (1995) J. Biol. Chem. 270, 7750-7756 [Abstract/Free Full Text]
  19. Rossie, S., and Catterall, W. A. (1987) J. Biol. Chem. 262, 12735-12744 [Abstract/Free Full Text]
  20. Murphy, B. J., and Catterall, W. A. (1992) J. Biol. Chem. 267, 16129-16134 [Abstract/Free Full Text]
  21. Luo, K., Hurley, T. R., and Sefton, B. M. (1991) Methods Enzymol. 201, 149-152 [Medline] [Order article via Infotrieve]
  22. Roskoski, R., Jr. (1983) Methods Enzymol. 99, 3-6 [Medline] [Order article via Infotrieve]
  23. Forn, J., and Greengard, P. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 5195-5199 [Abstract]
  24. Costa, M. R. C., and Catterall, W. A. (1984) J. Biol. Chem. 259, 8210-8218 [Abstract/Free Full Text]
  25. de Wit, R. J. W., Hoppe, J., Stec, W. J., Baraniak, J., and Jastorff, B. (1982) Eur. J. Biochem. 122, 95-99 [Abstract]
  26. Rothermel, J. D., Stec, W. J., Baraniak, J., Jastorff, B., and Parker Botelho, L. H. (1983) J. Biol. Chem. 258, 12125-12128 [Abstract/Free Full Text]
  27. Wu, W. C.-S., Walaas, S. I., Nairn, A. C., and Greengard, P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 5249-5253 [Abstract]
  28. Wang, J. K. T., Walaas, S. I., and Greengard, P. (1988) J. Neurosci. 8, 281-288 [Abstract]
  29. Costa, M. R. C., and Catterall, W. A. (1984) Cell. Mol. Neurobiol. 4, 291-297 [Medline] [Order article via Infotrieve]
  30. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., and Kirilovsky, J. (1991) J. Biol. Chem. 266, 15771-15781 [Abstract/Free Full Text]
  31. ffrench-Mullen, J. M. H., Danks, P., and Spence, K. T. (1994) J. Neurosci. 14, 1963-1977 [Abstract]
  32. Pearson, R. B., and Kemp, B. E. (1991) Methods Enzymol. 200, 62-81 [Medline] [Order article via Infotrieve]
  33. Dekker, L. V., and Parker, P. J. (1994) Trends Biochem. Sci. 19, 73-77 [CrossRef][Medline] [Order article via Infotrieve]
  34. Klauck, T. M., Faux, M. C., Labudda, K., Langeberg, L. K., Jaken, S., and Scott, J. D. (1996) Science 271, 1589-1592 [Abstract]
  35. Hashimoto, Y., and Soderling, T. R. (1989) J. Biol. Chem. 264, 16524-16529 [Abstract/Free Full Text]
  36. Quintaje, S. B., Church, D. J., Rebsamen, M., Valloton, M. B., Hemmings, B. A., and Lang, U. (1996) Biochem. Biophys. Res. Commun. 221, 539-547 [CrossRef][Medline] [Order article via Infotrieve]
  37. Coffey, E. T., Sihra, T. S., and Nicholls, D. G. (1993) J. Biol. Chem. 268, 21060-21065 [Abstract/Free Full Text]
  38. Chandler, L. J., and Leslie, S. W. (1989) J. Neurochem. 52, 1905-1912 [Medline] [Order article via Infotrieve]
  39. Barrie, A. P., Nicholls, D. G., Sanchez-Prieto, J., and Sihra, T. S. (1991) J. Neurochem. 57, 1398-1404 [Medline] [Order article via Infotrieve]
  40. Robinson, P. J. (1992) J. Biol. Chem. 267, 21637-21644 [Abstract/Free Full Text]
  41. Augustine, G. J., Charlton, M. P., and Smith, S. J. (1987) Annu. Rev. Neurosci. 10, 633-693 [CrossRef][Medline] [Order article via Infotrieve]
  42. Trimble, W. S., Linial, M., and Scheller, R. H. (1991) Annu. Rev. Neurosci. 14, 93-122 [CrossRef][Medline] [Order article via Infotrieve]
  43. Nicholls, D. G. (1993) Eur. J. Biochem. 212, 613-631 [Medline] [Order article via Infotrieve]
  44. Roach, P. J. (1991) J. Biol. Chem. 266, 14139-14142 [Abstract/Free Full Text]
  45. Guan, K.-L., and Butch, E. (1995) J. Biol. Chem. 270, 7197-7203 [Abstract/Free Full Text]
  46. Denu, J. M., Zhou, G., Wu, L., Zhao, R., Yuvaniyama, J., Saper, M. A., and Dixon, J. E. (1995) J. Biol. Chem. 270, 3796-3803 [Abstract/Free Full Text]
  47. Desdouits, F., Siciliano, J. C., Greengard, P., and Girault, J.-A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2682-2685 [Abstract]
  48. Bornancin, F., and Parker, P. J. (1996) Curr. Biol. 6, 1114-1123 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.