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Na+ Channel Regulation by Calmodulin Kinase II in Rat Cerebellar Granule Cells
Biochemical and Biophysical Research Communications 274, 394 –399 (2000) doi:10.1006/bbrc.2000.3145, available online at http://www.idealibrary.com on
Na ⫹ Channel Regulation by Calmodulin Kinase II in Rat Cerebellar Granule Cells Edmond Carlier, 1 Be´ne´dicte Dargent, Michel De Waard, and Franc¸ois Couraud Institut National de la Sante´ et de la Recherche Me´dicale U464, Laboratoire de Neurobiologie des Canaux Ioniques, Institut Jean Roche, Faculte´ de Me´decine Secteur Nord, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France
Received June 5, 2000
The effects of specific CaM kinase II inhibitors were investigated on Na ⴙ channels from rat cerebellar granule cells. A maximal effect of KN-62 was observed at 20 M and consisted of an 80% reduction of the peak Na ⴙ current after only a 10-min application. A hyperpolarizing shift of 8 mV in the steady-state inactivation was also observed. KN-04 (20 M), an inactive analog, had no detectable effect. KN-62 was however inactive on Na ⴙ currents recorded from Chinese hamster ovary cells expressing the type II A ␣ subunit. We have also analyzed the inhibitory effects of CaM kinase II 296 – 311 and CaM kinase II 281–309 peptides. Both peptides (75 M) induced a maximum peak Na ⴙ current reduction within 30 min. Under similar conditions, a truncated peptide CaM kinase II 284 –302 was ineffective. These results demonstrate that CaM kinase II acts as a modulator of Na ⴙ channel activity in cerebellar granule cells. © 2000 Academic Press Key Words: KN-62; KN-04; sodium currents; wholecell voltage clamp; cerebellar granule cells.
Voltage-sensitive sodium channels are essential for the initiation and propagation of action potentials by mediating a rapidly activated inward current that inactivates within a few milliseconds upon prolonged depolarization. Physiological modulation of voltagedependent Na ⫹ channels by cellular mechanisms has seldom been reported. Na ⫹ channels may be subject to regulation since previous studies have shown that ␣-subunits from nerve, muscle, and heart can be phosphorylated and several phosphorylation sites have indeed been identified (1–3). The activity of neuronal Abbreviations used: CaM kinase II, Ca 2⫹/calmodulin-dependent protein kinase II; DMSO, dimethyl sulfoxide; KN-04, N-[1-[Nmethyl-p-(5-isoquinolinesulfonyl)benzyl]-2-(4-phenylpiperazine)ethyl]5-isoquinolinesulfamide; KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)N-methyl-L-tyrosyl]-4-phenylpiperazine. 1 To whom correspondence should be addressed. Fax: (33) 4 91090506. E-mail: [email protected]. 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Na ⫹ channels can be modified by protein kinase C and/or protein kinase A phosphorylation. Thus, so far, protein kinase C activation reduces peak Na ⫹ current and slows the time course of inactivation (4, 5). Alterations in the voltage dependence of activation have also been observed (6). Protein kinase A reduces moderate currents flowing through rat brain type IIA ␣ channels expressed in Xenopus oocytes (7). Interestingly, a prominent reduction of peak Na ⫹ current by protein kinase A in neuronal cells (8) requires the concomitant activation of the channel by protein kinase C (9). On the other hand, Ma and collaborators (10) have demonstrated an increase in Na ⫹ current amplitude following pertussis toxin-sensitive G protein activation. Another likely route of phosphorylation is through the Ca 2⫹/calmodulin-dependent protein kinase (CaM kinase). The multifunctional CaM kinase II is particularly abundant in several regions of the brain where it may constitute up to 1–2% of total protein concentration (11). CaM kinase II has been implicated in various signaling pathways, and in particular may play a role in synaptic plasticity (12). In this study, we have analyzed the constitutive regulation of neuronal Na ⫹ channels by CaM-kinase II. Na ⫹ currents from cerebellar granule cells, maintained in primary culture, were recorded by the whole-cell voltage clamp technique and various potent kinase inhibitors were used to delineate the nature of the kinase regulation. The specific membranepermeable inhibitor, KN-62, and two inhibitory peptides derived from CaM kinase II, CMK 296 –311 and CMK 281–309, were used to study the effects of endogenous CaM kinase II on the Na ⫹ channels of cerebellar granule cells. KN-62 and the two inhibitory peptides induced a significant reduction in peak Na ⫹ current amplitude, whereas KN04, an inactive analog, and CMK 284 –302, a truncated CaM kinase II peptide, were without any effect. Our data favors the concept of a constitutive regulation of cerebellar Na⫹ channels by CaM kinase II.
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MATERIALS AND METHODS Primary granule cell cultures. Cerebellar granule cells from 7- to 8-day-old rat pups were mechanically dissociated and seeded at 2.5 ⫻ 10 6 cells on poly-L-ornithine-coated 35 mm diameter dishes (Costar). Granule cells were cultured in basal Dulbecco’s modified Eagle’s medium supplemented with 25 mM KCl, 2 mM glutamine (Sigma), 10% horse serum (GIBCO). Cells were incubated at 37°C in a humidified atmosphere, 10% CO 2 ⫹ 90% air. The dishes were transferred for patch-clamp experiments after 7–14 days in culture. CHO cell line culture. A Chinese hamster ovary cell line (CHOK1) transfected with NaIIA Na ⫹ channel cDNA (13) was kindly provided by Dr. W. Catterall (University of Washington, Seattle, WA). These cells were maintained in RPMI medium (GIBCO) supplemented with 5% fetal calf serum and incubated at 37°C in 5% CO 2. Electrophysiological recordings. The dishes containing the cell preparation studied were placed on the stage of an inverted microscope (Nikon Diaphot) equipped with Hoffman optics (modulation contrast, New York, NY) and viewed at a total magnification of 400⫻. Granular neurons were easily identified on the basis of their small size and round shape and most often appeared to be bipolar. Electrophysiological experiments were performed at room temperature (20 –22°C) with the single-electrode voltage clamp technique, using pipettes with a resistance that ranged from 2 to 4 M⍀ with our internal solution. The Na ⫹ gradient was reversed to eliminate variability in space clamp, allowing recordings of highly reproducible peak currents (5, 14). The external solution contained 90 mM choline Cl, 15 mM tetraethylammonium Cl, 1 mM MgCl 2, 1.5 mM CaCl 2, 1 mM KCl, 0.2 mM CdCl 2, 5 mM glucose, and 30 mM Hepes (pH adjusted to 7.3 with TMAOH). The internal solution contained 100 mM NaF, 30 mM NaCl, 20 mM CsF, and 5 mM Hepes (pH adjusted to 7.3 with CsOH). Whole-cell currents were recorded with an Axopatch 200A patch-clamp amplifier (Axon Instruments), low-pass filtered at 4 kHz with an 8 pole Bessel filter, and sampled at 20 kHz using a 12-bit ADC (Labmaster TM40, Scientific solution, Foster City, CA). Capacitance transients were cancelled and series resistance was compensated (⬎60%) using the internal voltage-clamp circuitry. Remaining capacitance and leak currents were subtracted from active currents using an on-line P/4 protocol (15). Data acquisition and analysis were controlled by pCLAMP6 software (Axon Instrument). The results are presented as mean ⫾ SEM, and significance of difference was determined by statistical t tests. Chemicals. The three peptides used in these experiments were diluted at a concentration of 75 M in the intracellular solution. Both CMK 296 –311 and CMK 281–309 peptides were purchased from LC Laboratories whereas the CMK 284 –302 peptide was synthesized by CIML (Marseille, France). KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-Nmethyl-L-tyrosyl]-4-phenylpiperazine) or KN-04 (N-[1-[N-methyl-p(5-isoquinolinesulfonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinolinesulfonamide) were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mM and bath applied after dilution in the extracellular recording medium. Bath applications of DMSO up to 1% had no measurable effects onto the Na ⫹ currents recorded from either cell types. KN-62 was purchased from LC Laboratories. KN-04 was purchased from Seikagaku corporation (Japan). All other reagents were of analytical grade and purchased from Sigma chemical company.
RESULTS KN-62 Inhibition of Na ⫹ Currents in Cerebellar Granule Cells To isolate Na ⫹ currents in cerebellar granule cells, whole cell recordings were performed in the presence of
CsCl (20 mM) and TEA-Cl (15 mM) to block K ⫹ currents and by adding 0.2 mM CdCl 2 to eliminate Ca 2⫹ currents. Figure 1A shows a voltage clamp recording of Na ⫹ currents from a representative cerebellar granule cell. In cells held at ⫺90 mV, I Na families were recorded by 45 ms depolarizing test pulses rising in 8 mV steps from ⫺60 to ⫹60 mV at a frequency of 0.25 Hz. The inverted sodium concentration gradient used in our experimental conditions resulted in outward Na ⫹ currents as a result of the change in driving force. As a first attempt to determine whether CaM kinase II regulates the voltage-dependent Na ⫹ channels, we tested the effects of the membrane-permeable CaM kinase II inhibitor, KN-62. KN-62 is a selective and potent CaM kinase II blocker that competes with calmodulin for its binding site on the enzyme (16). In the example illustrated in Fig. 1B, the extracellular application of 20 M KN-62 reduced the Na ⫹ current amplitude to about 30% of its original level. The drug mainly affected the peak Na ⫹ current amplitude but not the current kinetics. Thus, the inactivation kinetic was well described by a monoexponential fit and no apparent difference was observed in the decay time course (for example with a test pulse to ⫹ 12 mV; time constants of inactivation were ⫽ 1.26 ⫾ 0.16 ms for control vs ⫽ 1.33 ⫾ 0.11 ms after KN-62 application. NS at P ⬎ 0.05). In addition to reducing peak current amplitude, KN-62 also modified the voltage-dependence of inactivation of the Na ⫹ channel. The voltage-dependence of inactivation is described in Fig. 1C where currents during the test pulse were normalized to the largest outward current and plotted versus prepulse potential. These steady-state inactivation curves (n ⫽ 4) were determined with a 200-ms prepulse from ⫺110 mV to 0 mV in 10 mV steps, followed by a test pulse to ⫹40 mV. The values of half-inactivation (V 1/2) and slope (k) were ⫺72.08 ⫾ 0.32 mV and 6.73 ⫾ 0.28 mV for control condition and ⫺80.12 ⫾ 0.68 mV and 8.35 ⫾ 0.6 mV in the presence of 20 M KN-62, respectively. The slight 8 mV hyperpolarizing shift in inactivation appeared statistically significant. In contrast, we found no difference in the voltage-dependence of activation with half-activation potentials (V 1/2) and slopes (k) of 0.04 ⫾ 0.57 mV and 18.2 ⫾ 0.5 mV for control condition and ⫺3.55 ⫾ 0.38 mV and 17.55 ⫾ 0.5 mV after application of KN-62 (n ⫽ 4), respectively. The time course of KN-62-induced change in Na ⫹ channel properties was a function of the KN-62 concentration used. As indicated in Fig. 2, a bath application of 20 M KN-62 produced a maximal effect on the peak Na ⫹ current within 10 min. After this time, only 16 ⫾ 9.79% of the Na ⫹ remained (n ⫽ 4) compared to control. For a smaller concentration, such as 5 M, KN-62 induced a more progressive effect, that developed during the 30 min after KN-62 application. At this concentration, a larger fraction of 30.26 ⫾ 7.71% (n ⫽ 5) of the current remained after 30 min applica-
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tion. A larger block was however expected for longer applications. In contrast, application of 1.5 M KN-62 resulted in only a transient (10 min after application) and weaker effect (82.6 ⫾ 4.3% of control values, n ⫽ 3). Application of DMSO alone at a concentration of 1% was without any effect on the Na ⫹ current amplitude (n ⫽ 5). To check the specificity of KN-62 effect, we also used KN-04, a structural analog, that does not inhibit CaM kinase II activity. After an application of 10 min, 20 M KN-04 was ineffective on the Na ⫹ peak current amplitude (Figs. 1D and 1E) since no difference was observed with control values (101 ⫾ 5%, n ⫽ 5, NS at P ⬎ 0.05). Lack of Effect of KN-62 on Na ⫹ Currents from CNaIIA Cells We examined the effect of an application of 20 M KN-62 on Na ⫹ currents from CHO-KI cells by wholecell voltage clamp. This cell line expresses the Na ⫹ channel alphaIIA subunit alone. A 10-min application of KN-62 had no effect on the peak Na ⫹ current amplitude (93.18 ⫾ 13.8% of control current amplitude, n ⫽ 14, NS at P ⬎ 0.05, data not shown). Block of Cerebellar Granule Cell Na ⫹ Currents by CaM Kinase II Inhibitory Peptides
FIG. 1. The specific CaM kinase II inhibitor, KN-62, blocks Na ⫹ currents in cerebellar granule cells. Whole-cell voltage clamp experiments were carried out in cerebellar granule cells. (A, B) Trace current examples of a cell held at ⫺90 mV. Families of outward Na ⫹ currents were evoked by 8 mV depolarizing voltage steps from ⫺60 to ⫹60 mV before (A) and 10 min after 20 M KN-62 application (B). Note the important reduction in current amplitude induced by KN62. (C) Voltage dependence of inactivation (triangles) and activation (circles) of Na ⫹ currents recorded before (open symbols) and 10 min after (closed symbols) application of 20 M KN-62 (n ⫽ 8). Currents during the test pulse were normalized to the largest outward current. Inactivation was followed by applying 200 ms prepulses varying from ⫺110 mV to 0 mV in 10 mV steps before current induction by a pulse to 40 mV (n ⫽ 4). Activation curves are fitted to the Boltzmann equation 1/1 ⫹ exp[(V 1/2 ⫺ V m)/k], where V m is the test potential, V 1/2 is the half-activation potential and k is the slope factor. The inactivation curves are also fitted by a Boltzmann equation of the type 1/1 ⫹ exp[(V m ⫺ V 1/2)/k] where V m is the conditioning potential, V 1/2 is the half-inactivation potential, and k is the slope factor.
In a second set of experiments, we tested the effects of two CaM kinase II inhibitory peptides onto Na ⫹ currents by including these peptides into the microelectrode for cytoplasmic diffusion in granule cells. CMK 296 –311, a synthetic CaM-binding peptide, the sequence of which is based on the CaM-binding domain of CaM kinase II, has been shown to bind potently to CaM and inhibit the CaM-dependent activation of CaM kinase II (17). As depicted in Fig. 3A, the peak Na ⫹ currents, recorded at different times after breaking into the cell, showed a progressively stronger inhibition. After 30 min of cell perfusion by 75 M CMK 296 –311, the current Na ⫹ amplitude was reduced to 42 ⫾ 3.75% (n ⫽ 16) of its average control value. Under similar experimental conditions, we used a second type of inhibitory peptide, CMK 281–309, the sequence of which corresponds to a portion of the auto-inhibitory and calmodulin-binding domains of CaM kinase II. The results showed an even more progressive reduction in the peak Na ⫹ current amplitude than the one observed with CMK 296 –311 (Fig. 3A). This difference in kinetic of action may be due to a slower diffusion of the peptide
A 8 mV shift toward the hyperpolarizing direction of the inactivation curve was observed in presence of KN-62. (D, E) Under similar experimental conditions as described in (A) and (B), 20 M KN-04, an inactive analogue of KN-62, was ineffective after 10 min application.
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suggest a strong and constitutive regulation of Na ⫹ channels from cerebellar granule cell by CaM kinase II. KN-62 belongs to the isoquinolone sulfonamide series of protein kinase inhibitors (16, 18). In rat cerebellar granule cells, our main observation is that 5 to
FIG. 2. Concentration-dependent block of peak Na ⫹ current by KN-62. The change in the peak Na ⫹ current amplitude as a function of KN-62 application time was dependent of the dose used. Three concentrations of KN-62 were applied to the cells, 1.5 M (n ⫽ 4, filled triangles), 5 M (n ⫽ 5, filled squares) and 20 M (n ⫽ 4, filled circles), and the time course of KN-62 effect was analyzed up to 30 min. The maximal effect was obtained for 20 M KN-62 10 min after its application. With 5 and 20 M of KN-62, the decay of the current amplitude were best fitted with a single exponential component with a time constant of ⫽ 11.11 ⫾ 1.01 min and 3.61 ⫾ 0.43 min, respectively. DMSO (1%) concentration was used as control condition (n ⫽ 5, open circles).
owing to its larger sequence. However, a 30-min perfusion of CMK 281–309 resulted in a strong reduction in current amplitude, up to 43.1 ⫾ 5.2% (n ⫽ 7) of control currents. The amplitude of the effect of CMK 281–309 was thus similar to the one produced by CMK 296 –311. In contrast to KN-62, we found that the properties in voltage dependence of the Na ⫹ currents were not modified by the CMK inhibitor peptides. Figure 3B shows that activation and inactivation curves were indeed similar even after a 30 min application of CMK 296 –311 (n ⫽ 6). Finally, to control that the peptides did not act through mechanisms unrelated to their inhibition of CaM kinase II, we used the shorter peptide CMK 284 –302. As expected, we observed no variation in the peak Na ⫹ current recorded from cells perfused with 75 M CMK 284 –302 (Fig. 3A, n ⫽ 6). DISCUSSION The aim of the present study was to determine the effect of various CaM kinase II inhibitors onto the electrophysiological properties of Na ⫹ channels expressed in cerebellar granule neurons. We examined the ability of either KN-62 or two inhibitory peptides to modulate Na ⫹ currents. Taken together, our findings
FIG. 3. CMK 296 –311 and CMK 281–309, two inhibitory peptides of CaM kinase II, block peak Na ⫹ current. (A) CMK 296 –311, CMK 281–309, and CMK 284 –302 were diluted in the intracellular recording solution at a concentration of 75 M. The effects of the peptide on the peak Na ⫹ current amplitude were analyzed as a function of time after the start of the whole cell configuration. CMK 296 –311 (n ⫽ 16, filled circles) and CMK 281–309 (n ⫽ 7, filled triangles) induced 60% inhibition of the Na ⫹ current 30 min after intracellular dialysis. Under similar experimental conditions, the shorter peptide CMK 284 –302 (n ⫽ 6, filled squares) was ineffective at 75 M. The best fit of the data was obtained with a single exponential curve with time constants of ⫽ 18.08 ⫾ 1.89 min for CMK 296 –311 and ⫽ 22.64 ⫾ 5.84 min for CMK 281–309. (B) The voltage dependencies of activation and inactivation were not affected by peptides inhibitors of CaM kinase. With the usual conditions, no differences were observed between control cells (n ⫽ 6) recorded 30 min after breaking the membrane seal and cells (n ⫽ 6) recorded 30 min after dialysis of CMK 296 –311.
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20 M KN-62 strongly reduces the peak Na ⫹ current by about 80%, while 20 M KN-04, a structural and inactive analog of KN-62, is totally ineffective. The most likely explanation to our data is that Na ⫹ channels are constitutively activated by CaM kinase II phosphorylation in the basal state and that CaM kinase II inhibitors interfere with this activation. Na ⫹ channels are certainly likely molecular targets for regulation by kinases since it was found that they are substantially phosphorylated in the basal state in rat brain neurons (19). Additionally, Fukunaga and Soderling (20) have shown that basal cytoplasmic Ca 2⫹ levels, an estimated 30 to 60 nM in cerebellar granule cells, are sufficient to partially stimulate CaM kinase II activity in these cells. A chief argument to support the hypothesis of a basal phosphorylation of Na ⫹ channels by CaM kinase II is that KN-04 has no effect compared to KN-62. KN-04 is not an effective inhibitor of purified CaM Kinase II since it acts with a K i of 100 M compared to a K i of 0.1 M for KN-62. Indeed, it has been reported that 1 M KN-62 inhibits purified CaM Kinase II by over 90% whereas 10 M KN-04 causes only 20% inhibition (21). In our experiments, KN-04 at 20 M, a compound that is 100-fold less active in inhibiting purified CaM kinase II, does not show any effect. This result argues that CaM Kinase II regulates the activity of Na ⫹ channels by phosphorylating either the Na ⫹ channel directly or an associated protein. In addition to a reduction in peak current amplitude, we also observed a small shift (8 mV) of the steadystate inactivation curve towards the hyperpolarizing direction. This effect can also contribute to the observed decrease of the macroscopic Na ⫹ current. However, at ⫺90 mV, the potential at which were held the neurons in voltage-clamp, this leftward shift of the inactivation curve may only maximally contribute to 25% of the reduction in peak Na ⫹ current (Fig. 1). This percentage of inhibition is certainly insufficient to explain the 80% inhibition observed after KN-62 bath application. Also, reduction in peak current amplitude of the macroscopic Na ⫹ currents is also the major change that occurs as a result of protein kinase A or protein kinase C regulation of rat brain Na ⫹ type II channels (8, 9, 22). Also, the results reported herein with two specific peptide inhibitors of CaM Kinase II confirm the results obtained with KN-62. KN-62 competes with calmodulin for the calmodulin-binding site on the enzyme but does not inhibit the activity of auto-phosphorylated CaM kinase II (16). It is interesting to note that the inhibitory peptides used here, that correspond to either auto-inhibitory motifs or to a CaM binding domain (23), may also inhibit Na ⫹ currents. The somewhat smaller effect observed with the two peptides in comparison to the one obtained with KN-62, may be related to the capacity of each molecule to access its site of
action. The membrane-permeable KN-62 induces a maximal effect owing to a facilitated cell entry whereas the two peptides have to diffuse to their target following cell dialysis. The absence of any effect by the truncated peptide underlines the specificity of action of the two peptide inhibitors. It is also important to consider that, in cerebellar granule cells, the activity of protein phosphatases may reverse the phosphorylation occurring through the Ca 2⫹-independent form of CaM kinases II (24). In our experimental conditions, phosphatase activity has been limited by the use of fluor, which acts as an inhibitor of these enzymes. Whatever the mechanism whereby CaM kinase acts onto Na ⫹ channels, it is interesting to note that protein kinase A also regulates Na ⫹ channels, though in a complex way (25), and that CaM kinase II can inhibit type III adenylate cyclases (26). The results obtained here should also be correlated to the data presented by Dargent and collaborators (27) showing both a protective effect against veratridine neurotoxicity and a selective block of sodium influx by KN-62 in fetal cortical cells. The lack of KN-62 effect, observed in CNaIIA cells, seems to imply either that this CaM kinase II regulation is not related to the ␣ subunit itself or that CaM kinase II is absent in CHO cells. It should be noted that in cerebellar granule cells at least three ␣ isoforms, I, II, and III, and the  1 subunits are present (28). Future experiments will be aimed at identifying the specific sites of action of CaM kinase II on cerebellar granule cell Na ⫹ channels. REFERENCES
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Na ⫹ Channel Regulation by Calmodulin Kinase II in Rat Cerebellar Granule Cells Edmond Carlier, 1 Be´ne´dicte Dargent, Michel De Waard, and Franc¸ois Couraud Institut National de la Sante´ et de la Recherche Me´dicale U464, Laboratoire de Neurobiologie des Canaux Ioniques, Institut Jean Roche, Faculte´ de Me´decine Secteur Nord, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France
Received June 5, 2000
The effects of specific CaM kinase II inhibitors were investigated on Na ⴙ channels from rat cerebellar granule cells. A maximal effect of KN-62 was observed at 20 M and consisted of an 80% reduction of the peak Na ⴙ current after only a 10-min application. A hyperpolarizing shift of 8 mV in the steady-state inactivation was also observed. KN-04 (20 M), an inactive analog, had no detectable effect. KN-62 was however inactive on Na ⴙ currents recorded from Chinese hamster ovary cells expressing the type II A ␣ subunit. We have also analyzed the inhibitory effects of CaM kinase II 296 – 311 and CaM kinase II 281–309 peptides. Both peptides (75 M) induced a maximum peak Na ⴙ current reduction within 30 min. Under similar conditions, a truncated peptide CaM kinase II 284 –302 was ineffective. These results demonstrate that CaM kinase II acts as a modulator of Na ⴙ channel activity in cerebellar granule cells. © 2000 Academic Press Key Words: KN-62; KN-04; sodium currents; wholecell voltage clamp; cerebellar granule cells.
Voltage-sensitive sodium channels are essential for the initiation and propagation of action potentials by mediating a rapidly activated inward current that inactivates within a few milliseconds upon prolonged depolarization. Physiological modulation of voltagedependent Na ⫹ channels by cellular mechanisms has seldom been reported. Na ⫹ channels may be subject to regulation since previous studies have shown that ␣-subunits from nerve, muscle, and heart can be phosphorylated and several phosphorylation sites have indeed been identified (1–3). The activity of neuronal Abbreviations used: CaM kinase II, Ca 2⫹/calmodulin-dependent protein kinase II; DMSO, dimethyl sulfoxide; KN-04, N-[1-[Nmethyl-p-(5-isoquinolinesulfonyl)benzyl]-2-(4-phenylpiperazine)ethyl]5-isoquinolinesulfamide; KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)N-methyl-L-tyrosyl]-4-phenylpiperazine. 1 To whom correspondence should be addressed. Fax: (33) 4 91090506. E-mail: [email protected]. 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Na ⫹ channels can be modified by protein kinase C and/or protein kinase A phosphorylation. Thus, so far, protein kinase C activation reduces peak Na ⫹ current and slows the time course of inactivation (4, 5). Alterations in the voltage dependence of activation have also been observed (6). Protein kinase A reduces moderate currents flowing through rat brain type IIA ␣ channels expressed in Xenopus oocytes (7). Interestingly, a prominent reduction of peak Na ⫹ current by protein kinase A in neuronal cells (8) requires the concomitant activation of the channel by protein kinase C (9). On the other hand, Ma and collaborators (10) have demonstrated an increase in Na ⫹ current amplitude following pertussis toxin-sensitive G protein activation. Another likely route of phosphorylation is through the Ca 2⫹/calmodulin-dependent protein kinase (CaM kinase). The multifunctional CaM kinase II is particularly abundant in several regions of the brain where it may constitute up to 1–2% of total protein concentration (11). CaM kinase II has been implicated in various signaling pathways, and in particular may play a role in synaptic plasticity (12). In this study, we have analyzed the constitutive regulation of neuronal Na ⫹ channels by CaM-kinase II. Na ⫹ currents from cerebellar granule cells, maintained in primary culture, were recorded by the whole-cell voltage clamp technique and various potent kinase inhibitors were used to delineate the nature of the kinase regulation. The specific membranepermeable inhibitor, KN-62, and two inhibitory peptides derived from CaM kinase II, CMK 296 –311 and CMK 281–309, were used to study the effects of endogenous CaM kinase II on the Na ⫹ channels of cerebellar granule cells. KN-62 and the two inhibitory peptides induced a significant reduction in peak Na ⫹ current amplitude, whereas KN04, an inactive analog, and CMK 284 –302, a truncated CaM kinase II peptide, were without any effect. Our data favors the concept of a constitutive regulation of cerebellar Na⫹ channels by CaM kinase II.
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MATERIALS AND METHODS Primary granule cell cultures. Cerebellar granule cells from 7- to 8-day-old rat pups were mechanically dissociated and seeded at 2.5 ⫻ 10 6 cells on poly-L-ornithine-coated 35 mm diameter dishes (Costar). Granule cells were cultured in basal Dulbecco’s modified Eagle’s medium supplemented with 25 mM KCl, 2 mM glutamine (Sigma), 10% horse serum (GIBCO). Cells were incubated at 37°C in a humidified atmosphere, 10% CO 2 ⫹ 90% air. The dishes were transferred for patch-clamp experiments after 7–14 days in culture. CHO cell line culture. A Chinese hamster ovary cell line (CHOK1) transfected with NaIIA Na ⫹ channel cDNA (13) was kindly provided by Dr. W. Catterall (University of Washington, Seattle, WA). These cells were maintained in RPMI medium (GIBCO) supplemented with 5% fetal calf serum and incubated at 37°C in 5% CO 2. Electrophysiological recordings. The dishes containing the cell preparation studied were placed on the stage of an inverted microscope (Nikon Diaphot) equipped with Hoffman optics (modulation contrast, New York, NY) and viewed at a total magnification of 400⫻. Granular neurons were easily identified on the basis of their small size and round shape and most often appeared to be bipolar. Electrophysiological experiments were performed at room temperature (20 –22°C) with the single-electrode voltage clamp technique, using pipettes with a resistance that ranged from 2 to 4 M⍀ with our internal solution. The Na ⫹ gradient was reversed to eliminate variability in space clamp, allowing recordings of highly reproducible peak currents (5, 14). The external solution contained 90 mM choline Cl, 15 mM tetraethylammonium Cl, 1 mM MgCl 2, 1.5 mM CaCl 2, 1 mM KCl, 0.2 mM CdCl 2, 5 mM glucose, and 30 mM Hepes (pH adjusted to 7.3 with TMAOH). The internal solution contained 100 mM NaF, 30 mM NaCl, 20 mM CsF, and 5 mM Hepes (pH adjusted to 7.3 with CsOH). Whole-cell currents were recorded with an Axopatch 200A patch-clamp amplifier (Axon Instruments), low-pass filtered at 4 kHz with an 8 pole Bessel filter, and sampled at 20 kHz using a 12-bit ADC (Labmaster TM40, Scientific solution, Foster City, CA). Capacitance transients were cancelled and series resistance was compensated (⬎60%) using the internal voltage-clamp circuitry. Remaining capacitance and leak currents were subtracted from active currents using an on-line P/4 protocol (15). Data acquisition and analysis were controlled by pCLAMP6 software (Axon Instrument). The results are presented as mean ⫾ SEM, and significance of difference was determined by statistical t tests. Chemicals. The three peptides used in these experiments were diluted at a concentration of 75 M in the intracellular solution. Both CMK 296 –311 and CMK 281–309 peptides were purchased from LC Laboratories whereas the CMK 284 –302 peptide was synthesized by CIML (Marseille, France). KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-Nmethyl-L-tyrosyl]-4-phenylpiperazine) or KN-04 (N-[1-[N-methyl-p(5-isoquinolinesulfonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinolinesulfonamide) were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mM and bath applied after dilution in the extracellular recording medium. Bath applications of DMSO up to 1% had no measurable effects onto the Na ⫹ currents recorded from either cell types. KN-62 was purchased from LC Laboratories. KN-04 was purchased from Seikagaku corporation (Japan). All other reagents were of analytical grade and purchased from Sigma chemical company.
RESULTS KN-62 Inhibition of Na ⫹ Currents in Cerebellar Granule Cells To isolate Na ⫹ currents in cerebellar granule cells, whole cell recordings were performed in the presence of
CsCl (20 mM) and TEA-Cl (15 mM) to block K ⫹ currents and by adding 0.2 mM CdCl 2 to eliminate Ca 2⫹ currents. Figure 1A shows a voltage clamp recording of Na ⫹ currents from a representative cerebellar granule cell. In cells held at ⫺90 mV, I Na families were recorded by 45 ms depolarizing test pulses rising in 8 mV steps from ⫺60 to ⫹60 mV at a frequency of 0.25 Hz. The inverted sodium concentration gradient used in our experimental conditions resulted in outward Na ⫹ currents as a result of the change in driving force. As a first attempt to determine whether CaM kinase II regulates the voltage-dependent Na ⫹ channels, we tested the effects of the membrane-permeable CaM kinase II inhibitor, KN-62. KN-62 is a selective and potent CaM kinase II blocker that competes with calmodulin for its binding site on the enzyme (16). In the example illustrated in Fig. 1B, the extracellular application of 20 M KN-62 reduced the Na ⫹ current amplitude to about 30% of its original level. The drug mainly affected the peak Na ⫹ current amplitude but not the current kinetics. Thus, the inactivation kinetic was well described by a monoexponential fit and no apparent difference was observed in the decay time course (for example with a test pulse to ⫹ 12 mV; time constants of inactivation were ⫽ 1.26 ⫾ 0.16 ms for control vs ⫽ 1.33 ⫾ 0.11 ms after KN-62 application. NS at P ⬎ 0.05). In addition to reducing peak current amplitude, KN-62 also modified the voltage-dependence of inactivation of the Na ⫹ channel. The voltage-dependence of inactivation is described in Fig. 1C where currents during the test pulse were normalized to the largest outward current and plotted versus prepulse potential. These steady-state inactivation curves (n ⫽ 4) were determined with a 200-ms prepulse from ⫺110 mV to 0 mV in 10 mV steps, followed by a test pulse to ⫹40 mV. The values of half-inactivation (V 1/2) and slope (k) were ⫺72.08 ⫾ 0.32 mV and 6.73 ⫾ 0.28 mV for control condition and ⫺80.12 ⫾ 0.68 mV and 8.35 ⫾ 0.6 mV in the presence of 20 M KN-62, respectively. The slight 8 mV hyperpolarizing shift in inactivation appeared statistically significant. In contrast, we found no difference in the voltage-dependence of activation with half-activation potentials (V 1/2) and slopes (k) of 0.04 ⫾ 0.57 mV and 18.2 ⫾ 0.5 mV for control condition and ⫺3.55 ⫾ 0.38 mV and 17.55 ⫾ 0.5 mV after application of KN-62 (n ⫽ 4), respectively. The time course of KN-62-induced change in Na ⫹ channel properties was a function of the KN-62 concentration used. As indicated in Fig. 2, a bath application of 20 M KN-62 produced a maximal effect on the peak Na ⫹ current within 10 min. After this time, only 16 ⫾ 9.79% of the Na ⫹ remained (n ⫽ 4) compared to control. For a smaller concentration, such as 5 M, KN-62 induced a more progressive effect, that developed during the 30 min after KN-62 application. At this concentration, a larger fraction of 30.26 ⫾ 7.71% (n ⫽ 5) of the current remained after 30 min applica-
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tion. A larger block was however expected for longer applications. In contrast, application of 1.5 M KN-62 resulted in only a transient (10 min after application) and weaker effect (82.6 ⫾ 4.3% of control values, n ⫽ 3). Application of DMSO alone at a concentration of 1% was without any effect on the Na ⫹ current amplitude (n ⫽ 5). To check the specificity of KN-62 effect, we also used KN-04, a structural analog, that does not inhibit CaM kinase II activity. After an application of 10 min, 20 M KN-04 was ineffective on the Na ⫹ peak current amplitude (Figs. 1D and 1E) since no difference was observed with control values (101 ⫾ 5%, n ⫽ 5, NS at P ⬎ 0.05). Lack of Effect of KN-62 on Na ⫹ Currents from CNaIIA Cells We examined the effect of an application of 20 M KN-62 on Na ⫹ currents from CHO-KI cells by wholecell voltage clamp. This cell line expresses the Na ⫹ channel alphaIIA subunit alone. A 10-min application of KN-62 had no effect on the peak Na ⫹ current amplitude (93.18 ⫾ 13.8% of control current amplitude, n ⫽ 14, NS at P ⬎ 0.05, data not shown). Block of Cerebellar Granule Cell Na ⫹ Currents by CaM Kinase II Inhibitory Peptides
FIG. 1. The specific CaM kinase II inhibitor, KN-62, blocks Na ⫹ currents in cerebellar granule cells. Whole-cell voltage clamp experiments were carried out in cerebellar granule cells. (A, B) Trace current examples of a cell held at ⫺90 mV. Families of outward Na ⫹ currents were evoked by 8 mV depolarizing voltage steps from ⫺60 to ⫹60 mV before (A) and 10 min after 20 M KN-62 application (B). Note the important reduction in current amplitude induced by KN62. (C) Voltage dependence of inactivation (triangles) and activation (circles) of Na ⫹ currents recorded before (open symbols) and 10 min after (closed symbols) application of 20 M KN-62 (n ⫽ 8). Currents during the test pulse were normalized to the largest outward current. Inactivation was followed by applying 200 ms prepulses varying from ⫺110 mV to 0 mV in 10 mV steps before current induction by a pulse to 40 mV (n ⫽ 4). Activation curves are fitted to the Boltzmann equation 1/1 ⫹ exp[(V 1/2 ⫺ V m)/k], where V m is the test potential, V 1/2 is the half-activation potential and k is the slope factor. The inactivation curves are also fitted by a Boltzmann equation of the type 1/1 ⫹ exp[(V m ⫺ V 1/2)/k] where V m is the conditioning potential, V 1/2 is the half-inactivation potential, and k is the slope factor.
In a second set of experiments, we tested the effects of two CaM kinase II inhibitory peptides onto Na ⫹ currents by including these peptides into the microelectrode for cytoplasmic diffusion in granule cells. CMK 296 –311, a synthetic CaM-binding peptide, the sequence of which is based on the CaM-binding domain of CaM kinase II, has been shown to bind potently to CaM and inhibit the CaM-dependent activation of CaM kinase II (17). As depicted in Fig. 3A, the peak Na ⫹ currents, recorded at different times after breaking into the cell, showed a progressively stronger inhibition. After 30 min of cell perfusion by 75 M CMK 296 –311, the current Na ⫹ amplitude was reduced to 42 ⫾ 3.75% (n ⫽ 16) of its average control value. Under similar experimental conditions, we used a second type of inhibitory peptide, CMK 281–309, the sequence of which corresponds to a portion of the auto-inhibitory and calmodulin-binding domains of CaM kinase II. The results showed an even more progressive reduction in the peak Na ⫹ current amplitude than the one observed with CMK 296 –311 (Fig. 3A). This difference in kinetic of action may be due to a slower diffusion of the peptide
A 8 mV shift toward the hyperpolarizing direction of the inactivation curve was observed in presence of KN-62. (D, E) Under similar experimental conditions as described in (A) and (B), 20 M KN-04, an inactive analogue of KN-62, was ineffective after 10 min application.
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suggest a strong and constitutive regulation of Na ⫹ channels from cerebellar granule cell by CaM kinase II. KN-62 belongs to the isoquinolone sulfonamide series of protein kinase inhibitors (16, 18). In rat cerebellar granule cells, our main observation is that 5 to
FIG. 2. Concentration-dependent block of peak Na ⫹ current by KN-62. The change in the peak Na ⫹ current amplitude as a function of KN-62 application time was dependent of the dose used. Three concentrations of KN-62 were applied to the cells, 1.5 M (n ⫽ 4, filled triangles), 5 M (n ⫽ 5, filled squares) and 20 M (n ⫽ 4, filled circles), and the time course of KN-62 effect was analyzed up to 30 min. The maximal effect was obtained for 20 M KN-62 10 min after its application. With 5 and 20 M of KN-62, the decay of the current amplitude were best fitted with a single exponential component with a time constant of ⫽ 11.11 ⫾ 1.01 min and 3.61 ⫾ 0.43 min, respectively. DMSO (1%) concentration was used as control condition (n ⫽ 5, open circles).
owing to its larger sequence. However, a 30-min perfusion of CMK 281–309 resulted in a strong reduction in current amplitude, up to 43.1 ⫾ 5.2% (n ⫽ 7) of control currents. The amplitude of the effect of CMK 281–309 was thus similar to the one produced by CMK 296 –311. In contrast to KN-62, we found that the properties in voltage dependence of the Na ⫹ currents were not modified by the CMK inhibitor peptides. Figure 3B shows that activation and inactivation curves were indeed similar even after a 30 min application of CMK 296 –311 (n ⫽ 6). Finally, to control that the peptides did not act through mechanisms unrelated to their inhibition of CaM kinase II, we used the shorter peptide CMK 284 –302. As expected, we observed no variation in the peak Na ⫹ current recorded from cells perfused with 75 M CMK 284 –302 (Fig. 3A, n ⫽ 6). DISCUSSION The aim of the present study was to determine the effect of various CaM kinase II inhibitors onto the electrophysiological properties of Na ⫹ channels expressed in cerebellar granule neurons. We examined the ability of either KN-62 or two inhibitory peptides to modulate Na ⫹ currents. Taken together, our findings
FIG. 3. CMK 296 –311 and CMK 281–309, two inhibitory peptides of CaM kinase II, block peak Na ⫹ current. (A) CMK 296 –311, CMK 281–309, and CMK 284 –302 were diluted in the intracellular recording solution at a concentration of 75 M. The effects of the peptide on the peak Na ⫹ current amplitude were analyzed as a function of time after the start of the whole cell configuration. CMK 296 –311 (n ⫽ 16, filled circles) and CMK 281–309 (n ⫽ 7, filled triangles) induced 60% inhibition of the Na ⫹ current 30 min after intracellular dialysis. Under similar experimental conditions, the shorter peptide CMK 284 –302 (n ⫽ 6, filled squares) was ineffective at 75 M. The best fit of the data was obtained with a single exponential curve with time constants of ⫽ 18.08 ⫾ 1.89 min for CMK 296 –311 and ⫽ 22.64 ⫾ 5.84 min for CMK 281–309. (B) The voltage dependencies of activation and inactivation were not affected by peptides inhibitors of CaM kinase. With the usual conditions, no differences were observed between control cells (n ⫽ 6) recorded 30 min after breaking the membrane seal and cells (n ⫽ 6) recorded 30 min after dialysis of CMK 296 –311.
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20 M KN-62 strongly reduces the peak Na ⫹ current by about 80%, while 20 M KN-04, a structural and inactive analog of KN-62, is totally ineffective. The most likely explanation to our data is that Na ⫹ channels are constitutively activated by CaM kinase II phosphorylation in the basal state and that CaM kinase II inhibitors interfere with this activation. Na ⫹ channels are certainly likely molecular targets for regulation by kinases since it was found that they are substantially phosphorylated in the basal state in rat brain neurons (19). Additionally, Fukunaga and Soderling (20) have shown that basal cytoplasmic Ca 2⫹ levels, an estimated 30 to 60 nM in cerebellar granule cells, are sufficient to partially stimulate CaM kinase II activity in these cells. A chief argument to support the hypothesis of a basal phosphorylation of Na ⫹ channels by CaM kinase II is that KN-04 has no effect compared to KN-62. KN-04 is not an effective inhibitor of purified CaM Kinase II since it acts with a K i of 100 M compared to a K i of 0.1 M for KN-62. Indeed, it has been reported that 1 M KN-62 inhibits purified CaM Kinase II by over 90% whereas 10 M KN-04 causes only 20% inhibition (21). In our experiments, KN-04 at 20 M, a compound that is 100-fold less active in inhibiting purified CaM kinase II, does not show any effect. This result argues that CaM Kinase II regulates the activity of Na ⫹ channels by phosphorylating either the Na ⫹ channel directly or an associated protein. In addition to a reduction in peak current amplitude, we also observed a small shift (8 mV) of the steadystate inactivation curve towards the hyperpolarizing direction. This effect can also contribute to the observed decrease of the macroscopic Na ⫹ current. However, at ⫺90 mV, the potential at which were held the neurons in voltage-clamp, this leftward shift of the inactivation curve may only maximally contribute to 25% of the reduction in peak Na ⫹ current (Fig. 1). This percentage of inhibition is certainly insufficient to explain the 80% inhibition observed after KN-62 bath application. Also, reduction in peak current amplitude of the macroscopic Na ⫹ currents is also the major change that occurs as a result of protein kinase A or protein kinase C regulation of rat brain Na ⫹ type II channels (8, 9, 22). Also, the results reported herein with two specific peptide inhibitors of CaM Kinase II confirm the results obtained with KN-62. KN-62 competes with calmodulin for the calmodulin-binding site on the enzyme but does not inhibit the activity of auto-phosphorylated CaM kinase II (16). It is interesting to note that the inhibitory peptides used here, that correspond to either auto-inhibitory motifs or to a CaM binding domain (23), may also inhibit Na ⫹ currents. The somewhat smaller effect observed with the two peptides in comparison to the one obtained with KN-62, may be related to the capacity of each molecule to access its site of
action. The membrane-permeable KN-62 induces a maximal effect owing to a facilitated cell entry whereas the two peptides have to diffuse to their target following cell dialysis. The absence of any effect by the truncated peptide underlines the specificity of action of the two peptide inhibitors. It is also important to consider that, in cerebellar granule cells, the activity of protein phosphatases may reverse the phosphorylation occurring through the Ca 2⫹-independent form of CaM kinases II (24). In our experimental conditions, phosphatase activity has been limited by the use of fluor, which acts as an inhibitor of these enzymes. Whatever the mechanism whereby CaM kinase acts onto Na ⫹ channels, it is interesting to note that protein kinase A also regulates Na ⫹ channels, though in a complex way (25), and that CaM kinase II can inhibit type III adenylate cyclases (26). The results obtained here should also be correlated to the data presented by Dargent and collaborators (27) showing both a protective effect against veratridine neurotoxicity and a selective block of sodium influx by KN-62 in fetal cortical cells. The lack of KN-62 effect, observed in CNaIIA cells, seems to imply either that this CaM kinase II regulation is not related to the ␣ subunit itself or that CaM kinase II is absent in CHO cells. It should be noted that in cerebellar granule cells at least three ␣ isoforms, I, II, and III, and the  1 subunits are present (28). Future experiments will be aimed at identifying the specific sites of action of CaM kinase II on cerebellar granule cell Na ⫹ channels. REFERENCES
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