Calcium/Calmodulin-dependent Protein Kinase II Phosphorylates and Regulates the Drosophila Eag Potassium Channel*

, Modulation of neuronal excitability is believed to be an important mechanism of plasticity in the nervous system. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been postulated to regulate the ether a` go-go ( eag ) potassium channel in Drosophila . Inhibition of CaMKII and mutation of the eag gene both cause hyperexcitability at the larval neuromuscular junction (NMJ) and memory formation defects in the adult. In this study, we identify a single site, threonine 787, as the major CaMKII phosphorylation site in Eag. This site can be phosphorylated by CaMKII both in a heterologous cell system and in vivo at the larval NMJ. Expression of Eag in Xenopus oocytes was used to assess the function of phosphorylation. Injection of either a specific CaMKII inhibitor peptide or lavendustin C, another CaMKII inhibitor, reduced Eag current amplitude acutely. Mutation of threonine 787 to alanine also reduced amplitude. Moreover, both CaMKII inhibition and the alanine mutation accelerated inactivation. The reduction in current amplitudes and the accelerated inactivation of de-phosphorylated Eag channels would result in decreased outward potassium currents and hyperexcitability at presynaptic terminals and, thus, are consistent with the NMJ phenotype observed when CaMKII is inhibited. These results show that Escherichia coli PR745 (New England Biolabs) transformed with the appropriate plasmid was diluted at 1:100 in 1 liter of LB/carbenicillin and grown at 37 °C fo r 3 h oruntil A 600 (cid:2) 0.8. Protein expression was induced by adding isopropyl-1-thio- (cid:1) - D galactopyranoside (Promega) to a final concentration of 0.1 m M . The extracellular 140 m M m M KCl, m M MgCl , m M (pH to An extracel- lular high in K was used to enhance tail currents during measurements of the voltage dependence of this 100 m M KCl, 1.8 m M CaCl , 1 m M MgCl 2 , m M 2 and of amplitude measurements tion and measurements

Numerous studies have suggested that ion channels are an important class of neuronal targets for protein kinases. Modulation of potassium channel activity by kinases has key importance in regulation of neuronal excitability and synaptic plasticity (1). The ether-à -go-go (eag) gene in Drosophila encodes a voltage-gated potassium channel that has a C-terminal cytoplasmic domain with homology to cyclic nucleotide-gated channels (2,3). This channel is the founding member of a superfamily of vertebrate and invertebrate genes, which include eag, elk, and erg subfamilies (4). In flies, mutation of the eag gene causes shaking after exposure to ether (5) and hyperexcitability at the NMJ 1 (6). These phenotypes have been demonstrated with both hypomorphic and null alleles of eag (see, for example, the NMJ phenotypes of eag 1 , a hypomorph, and eag sc29 , a null, in Ref. 7). In humans, mutation of the HERG gene is one cause of long QT syndrome (8).
Genetic interactions between Drosophila calcium/calmodulindependent protein kinase II (CaMKII) and Eag have suggested the hypothesis that Eag is a downstream target for CaMKIImediated modulation of neuronal function (7). Inhibition of CaMKII by expression of a kinase-specific autoinhibitory domain peptide produced supernumerary excitatory junctional potentials at the third instar NMJ. This excitability defect was similar in many respects to that of eag mutants, and expression of the inhibitor on an eag background produced nonadditive effects on excitability. Both expression of inhibitory peptide and eag mutations blocked memory formation of courtship conditioning (7). These data suggested that CaMKII and Eag may function in a common pathway that regulates neuronal plasticity.
To investigate the direct interaction of Eag and CaMKII, we have identified a CaMKII phosphorylation site on Eag. We find that Eag is expressed in both the axon and the terminal boutons of motor neurons, where it is phosphorylated in vivo. We also present evidence that phosphorylation of this site can modulate channel function. culture was allowed to grow at 37°C for another 2 h. Cells were harvested by centrifugation at 5,000 ϫ g for 10 min.
The protocol for purification was modified from the manufacturer instructions from the glutathione-Sepharose-4B column (Amersham Biosciences). To purify the fusion proteins, cell pellets were resuspended in 40 ml of cold lysis buffer (20 mM Tris, pH 8, 100 mM NaCl, 1 mM EDTA, 10 g/ml aprotinin, 10 g/ml pepstatin, 10 g/ml leupeptin, 10 g/ml trypsin inhibitor, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 M microcystin-LR, and 1 mM dithiothreitol). 0.5 mg/ml lysozyme (Sigma) was added, and the mixture was incubated on ice for 30 min. Triton X-100 was added to a final concentration of 1%, and the mixture was sonicated at maximum setting three times (30 s each). After spinning at 10,000 ϫ g for 10 min, the supernatant was removed to a 50-ml tube. Glutathione-Sepharose-4B was prepared as instructed and equilibrated with the lysis buffer to make a 50% suspension. 2 ml of the bead suspension was added to the supernatant, and the mixture was rotated at 4°C for periods ranging from 2 h to overnight. The beads were spun down at low speed for 3 min in a clinical centrifuge at 4°C and washed three times (20 min each) by rotating gently, first with 40 ml of lysis buffer plus 1% Triton, second with 40 ml of lysis buffer plus 400 mM NaCl, and third with 40 ml of lysis buffer only. After each wash, beads were collected by spinning. The fusion proteins were eluted in four fractions of 2 ml each of freshly made elution buffer (50 mM Tris, pH 8.0, 10 mM reduced glutathione from Sigma) by rotating for 30 min at 4°C. Fractions were pooled and concentrated in a Centriprep column (Amicon) with a molecular mass cut-off of 30 kDa. Fusion proteins were snap-frozen on dry ice and stored at Ϫ20°C.
Anti-Eag Antibody-Generation of polyclonal rabbit antibodies employed standard procedures (9) at the Foster Animal Facility (Brandeis University, Waltham, MA). GST-Eag-N (250 g) fusion protein was mixed with Freund's adjuvant and injected into rabbits subcutaneously. Sera were screened against the antigen using dot blots and Western blots. Purification of anti-Eag antibodies was carried out according to Ref. 9; HIS-Eag-N was bound to a Ni 2ϩ -nitrilotriacetic acid column and washed with 10 volumes of Tris buffer (10 mM Tris-HCl, pH 7.5). Serum (in 25-ml batches) was loaded several times at a rate of 20 ml/h. The column was washed with 10 volumes of Tris buffer, 20 volumes of Tris buffer plus 500 mM NaCl, and 10 volumes of Tris buffer, pH 8.8. The column was eluted with 10 ml of tetraethylamine, pH 11.5, and the eluate neutralized with 1 ml of Tris buffer, pH 8. Eluates were pooled, dialyzed against PBS, and then concentrated.
Anti-Thr(P)-787 Antibody-A peptide corresponding to Eag amino acid sequence 784 -792 (NH 2 -CRQDpT787IDEGG, chemically phosphorylated at Thr-787) was synthesized (Alpha Diagnostic, Inc). The Nterminal cysteine was added to facilitate conjugation onto an agarose support. The peptide was conjugated to keyhole limpet hemocyanin as instructed (Pierce, Imject activated immunogen conjugation kit or Imject maleimide-activated keyhole limpet hemocyanin) for immunization (250 g of protein/rabbit/injection). For purification, the phosphopeptide was immobilized on a cross-linked agarose support (Pierce, SulfoLink kit) and the antibodies purified according to the manufacturer's instructions.
Phosphorylation of Fusion Proteins-GST fusion proteins were phosphorylated in an assay reaction of 50 l containing 50 mM PIPES, pH 7.0, 15 mM MgCl 2 , 1 mM CaCl 2 , 10 g/ml calmodulin, and either 50 M ATP or 50 M [␥-32 P]ATP (1 Ci/mmol). Reactions were run at 30°C. Following appropriate incubations, 25 l of SDS sample loading buffer (9% SDS, 25% glycerol, 0.186 M Tris pH 8.9, 5% 2-mercaptoethanol) was added to the reaction mixture and heated at 100°C for 3 min. Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting or exposed to film if radioactive.
cDNA Constructs-Eag cDNAs were cloned into pCS2ϩMT (10) for transfection of mammalian tsA201 cells and into pEGFP (CLONTECH) to produce GFP fusion proteins.
Transfection of tsA201 Cells and Eag Immunoprecipitation-tsA201 cells were grown at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% Cosmic calf serum (HyClone) in a humidified 95% O 2 and 5% CO 2 incubator. Cells were seeded at 10 6 cells/100-mm dish and transfected with DNA constructs 24 h later, using the calcium phosphate method (11). 15 g of Eag DNA and 7.5 g of CaMKII DNA were used per plate. DNA precipitates were removed 16 h after transfection, and the cells were allowed to grow for another 36 -40 h. Cells were harvested by pipetting in 1 ml/plate solubilization buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 50 mM NaF, 1 mM NaVO 3 , 1 mM EDTA, 1% CHAPS, 10 g/ml aprotinin, 10 g/ml pepstatin, 10 g/ml leupeptin, 10 g/ml trypsin inhibitor, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 1 M microcystin-LR). Cells were solubilized in microcentrifuge tubes by rotating at 4°C for 30 min and then spun for 15 min at 14,000 ϫ g. Supernatants were transferred to new tubes. The protein concentrations were determined by the Bradford method (Bio-Rad). Extracts containing 300 g of total protein were incubated with an anti-Eag antibody overnight at 4°C, and the immunocomplex was precipitated by incubating with protein A beads (Santa Cruz). After beads were washed three times with the solubilization buffer, proteins were eluted by boiling in loading buffer and separated by SDS-PAGE.
Immunoblotting-Proteins or cell extracts were separated by SDS-PAGE, transferred to nitrocellulose electrophoretically, and probed with antibodies as previously described (12). Affinity-purified anti-Eag was used at 1:500, and affinity-purified anti-Thr(P)-787 was used at 1:100. Relative intensity of signals was determined by densitometry using the Bio-Rad Gel Doc software.
Immunocytochemistry of Transfected Cells-tsA201 cells were grown on acid-washed, poly-D-lysine-coated glass coverslips (Fisher). After transfection, cells were washed twice with PBS (138 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 ) and fixed with 4% paraformaldehyde in PBS for 15 min. Cells were permeabilized by incubating with 0.1% Triton X-100 in PBS for 5 min at room temperature and washed three times with PBS (5 min each). Cells were then incubated with affinity-purified anti-Thr(P)-787 antibody (1:50) in PBS with 0.05% Tween 20 plus 10% normal goat serum for 60 min at 37°C. After washing five times, the cells were incubated with 1:200 antirabbit rhodamine-conjugated secondary antibody (Jackson Immunological Laboratory) for 30 min at 37°C. After five washes, the coverslips were mounted onto slides. The samples were examined using confocal microscopy (Bio-Rad 600 system).
Drosophila Stocks-Lines with mutations in the eag gene (eag 1 and eag sc29 ) were a gift of Barry Ganetzky (University of Wisconsin, Madison, WI). The eag 1 allele encodes a protein with a truncated C terminus 2 and is a hypomorph, whereas the eag sc29 mutant is a null for the full-length mRNA (13), and there is no detectable full-length protein in head extracts (data not shown). The pan-neural GAL4 driver line C155 (14) was used to drive transgene expression in fly neurons. This line contains a GAL4 gene inserted into the elav locus and expresses in all neurons (15). UAS-CaMKII-T287D was made as described (16). This transgene encodes a constitutively active form of Drosophila CaMKII (17).
Preparation and Phosphorylation of Fly Head Extracts-Canton-S flies were collected and quickly frozen in liquid N 2 . Heads were separated from bodies with a sizing sieve. Approximately 250 heads were homogenized in the solubilization buffer and spun for 10 min at 4°C. The supernatant was divided into four aliquots and mixed with CaMKII, phosphorylation assay reagents, and [␥-32 P]ATP. Calciumstimulated activity was measured in an assay reaction containing 50 mM PIPES, pH 7.0, 15 mM MgCl 2 , 1 mM CaCl 2 , 10 g/ml calmodulin, and 50 M [␥-32 P]ATP. In control reactions, 0.5 mM EGTA was used and CaCl 2 and CaM were omitted. CaMKII (R3 isoform) was purified as described from transfected COS cells (18). The reactions were allowed to proceed at 30°C for 10 min and terminated by adding 200 l of solubilization buffer and 5 l of the anti-Eag antibody. Immunoprecipitation was done by rotating tubes for 2 h at 4°C. The Eag-IgG complex was pulled down with protein A beads. The beads were washed, and proteins were boiled off in SDS sample loading buffer and separated on SDS gels. Gels were dried and subjected to autoradiography.
Immunohistochemistry of Third Instar Larvae-Male third instar larvae were immobilized in a chamber cut out on a Sylgard-covered glass slide with insect pins (Fine Science Tool, Inc.) and cut open along their dorsal surface with Aesculap Micro Scissors. Gut and brain were removed. Larval body wall muscles were dissected in low calcium saline (5 mM HEPES, pH 7.2, 128 mM NaCl, 2 mM KCl, 35.5 mM sucrose, 4 mM MgCl 2 , 2 mM EGTA) and fixed in PBS with 4% paraformaldehyde for 30 min. Preparations were washed three times (15 min each) with PBT (PBS, 0.3% Triton X-100, 0.1% bovine serum albumin) and incubated in 1:50 affinity-purified anti-Thr(P)-787 in PBT overnight at 4°C, followed by staining with a rhodamine-conjugated anti-rabbit IgG secondary antibody (Cappel, 1:200, 60 min at 37°C). The samples were washed three times with PBT and mounted using Vectorshield (Vector Laboratories).
Electrophysiology-For expression in Xenopus oocytes, SphI sites flanking the Thr-787 phosphorylation site were used to subclone singlesite mutations into the pGH19-eag construct (12). Plasmids were linearized using NotI and capped RNAs transcribed in vitro using T7 RNA polymerase according to manufacturer instructions (Message Machine, Ambion). RNA concentrations were quantified with spectrophotometric readings. Stage V-VI oocytes were defolliculated by incubation in Ca 2ϩfree OR2 solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 5 mM Hepes, pH to 7.6 with NaOH) containing 2 mg/ml collagenase (Sigma, type 1A) for 1-3 h with gentle agitation. Oocytes were injected with 0.1-0.2 ng of RNA/oocyte and were maintained in L-15 medium (containing: 50% L-15 (Invitrogen), 15 mM Hepes, 50 mg/ml gentamycin, and 5 mg/ml bovine serum albumin, pH to 7.4 with NaOH) at 18°C for 2-5 days. Recordings were performed using a Turbo TEC-10C amplifier (NPI Electronics) and pCLAMP8 software (Axon Instruments). The extracellular recording solution typically contained 140 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 10 mM Hepes (pH to 7.1 with NaOH). An extracellular solution high in K ϩ was used to enhance tail currents during measurements of the voltage dependence of activation; this solution contained 100 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5 mM Hepes (pH 7.4 with KOH). Pipettes were filled with 2 M KCl and had resistances of 0.3-0.6 megohms. Experiments were performed at room temperature (19 -22°C).
Unless otherwise noted, amplitude measurements refer to the peak currents observed during test pulses to the indicated voltages. Activation and inactivation time constants were determined by fitting traces (excluding capacitative transients) with two exponentials and a steady state. Conductance-voltage (G-V) relations were obtained from measurements of instantaneous tails currents. Curves were normalized by the maximum conductance and fit with a Boltzmann distribution. Measurements were statistically compared using unpaired Student's t tests with the Welch correction for unequal variances.
CaMKII autoinhibitory peptide (CaMKII inhibitor 281-301, Calbiochem) and lavendustin C (Calbiochem) were dissolved in water and Me 2 SO, respectively. Equal volumes of the stock solutions or their corresponding buffers were injected using a Nanoject (Drummond) to obtain the final concentrations noted. Oocyte volume was estimated assuming a diameter of 1 mm.

Native Eag Is a Substrate for CaMKII-Previous work had
shown that CaMKII could phosphorylate a bacterially produced fragment of Eag in vitro (7). To determine whether native Eag proteins in Drosophila were substrates for CaMKII, we prepared Canton-S fly head extracts. Radioactive ATP, purified CaMKII, Ca 2ϩ /CaM, and/or EGTA were added and the mixture incubated for 10 min. After the phosphorylation reaction was complete, Eag was immunoprecipitated. Fig. 1 shows 32 P incorporation into immunoprecipitated Eag protein visualized by autoradiography. In the presence of Ca 2ϩ /CaM and exogenous pure CaMKII, Eag proteins from Canton-S fly heads were phosphorylated. The Eag band was not seen when either Ca 2ϩ /CaM or CaMKII or both were absent in the reactions, suggesting that the phosphorylation was Ca 2ϩ /CaM-and CaMKII-dependent.
Mapping of the CaMKII Phosphorylation Site on Eag in Vitro-Eag is a transmembrane protein with cytoplasmic N and C termini. To localize the site(s) of Eag phosphorylation, we made GST fusion proteins containing both cytoplasmic do-mains. Fig. 2A shows a schematic diagram of Eag. GST-Eag-N is a 45-kDa fusion protein containing the N terminus of Eag from amino acid 44 to 210. This protein includes three CaMKII consensus sites (RXX(S/T)) at Thr-154, Thr-182, and Ser-188. GST-Eag-C1 is a 54-kDa fusion protein containing the C terminus of Eag from amino acid 556 -802. This fusion protein includes three CaMKII consensus sites at Ser-576, Thr-655, and Thr-787. Fig. 2B shows phosphorylation of GST, GST-Eag-N, and GST-Eag-C1 using purified Drosophila CaMKII, radioactive ATP, and Ca 2ϩ /CaM or EGTA. Phosphate incorporation into the fusion protein substrates was assessed by autoradiography. Only the C1 fragment was a substrate. Neither GST alone or GST-Eag-N was able to incorporate phosphate. CaMKII autophosphorylation is not seen on these gels because of the small amount of kinase used in the experiment.
To determine which amino acid residues in the Eag C terminus were CaMKII phosphorylation sites, we mutated all three consensus sites of GST-Eag-C1 to alanine by site-directed mutagenesis. In vitro phosphorylation assays were carried out using the purified Eag fragments. Fig. 3A shows the result from one such experiment. When the GST-Eag-C1 (wild type); single mutants S576A, T655A, and T787A; and double mutants T655A/T787A and S576A/T787A were subjected to phosphorylation by CaMKII, only C1-WT, S576A, and T655A were phosphorylated. Because none of the mutant proteins containing T787A were substrates for CaMKII, it suggested that Thr-787 was the major site of CaMKII phosphorylation in the proximal C terminus under these assay conditions.
GST-Eag-C2 and GST-Eag-C2-T787A were generated to test the possibility that there were additional sites in the C termi- nus. GST-Eag-C2 is a 95-kDa fusion protein containing the entire Eag C terminus from 556 to 1175. There are two additional CaMKII consensus sites, Thr-873 and Ser-1041, in this fusion protein compared with Eag-C1. Ser-1041 is also a consensus phosphorylation site for PKA, the only such site in Eag. Fig. 3B shows CaMKII phosphorylation of GST-Eag-C2 and GST-Eag-C2-T787A proteins. GST-Eag-C2 and its degradation products incorporated phosphate, whereas GST-Eag-C2-T787A was not phosphorylated at all, even at long incubation times. We conclude that Thr-787 of Eag is the only site that is phosphorylated by CaMKII in vitro under these assay conditions.
Threonine 787 of Eag Is Phosphorylated in Transfected tsA201 Cells-To allow us to determine whether Thr-787 was a site for CaMKII phosphorylation in the intact channel in vivo, we raised a phosphospecific antibody to this residue. Fig. 4A shows that the affinity-purified antibody recognized the phosphorylated GST-Eag-C2 but not the unphosphorylated form. GST-Eag-C2-T787A was not recognized even after kinase treatment.
The mammalian tsA201 cell line has been used to express potassium channel proteins at relatively high levels (19). We used anti-Thr(P)-787 to probe the phosphorylation state of Eag in tsA201 cells, which had been transiently transfected with Myc-tagged Eag-WT or Eag-T787A cDNA. To test the ability of CaMKII to phosphorylate Eag, a cDNA encoding a constitutively active form of CaMKII (CaMKII-T287D) was cotransfected. Eag proteins in cell extracts were immunoprecipitated with the anti-Eag antibody and analyzed by immunoblotting (Fig. 4B). Equal amounts of Eag were precipitated from each cell extract, as shown by Western blots using an antibody that recognizes the Myc epitope tag present on the channel. When the same immunoprecipitations were probed with the anti-Thr(P)-787 phosphospecific antibody, there was a 13-fold increase in the immunoreactivity in cells cotransfected with Eag-WT and CaMKII-T287D, compared with those transfected with Eag-WT alone. The basal signal seen with the wild type alone was probably a result of its phosphorylation by endogenous kinase. We and others (20) have observed a low level of CaMKII activity in this cell line. There was no signal detected in cells transfected with the mutant Myc-Eag-T787A, even in the presence of the constitutively active CaMKII.
Thr-787 can also be shown to be an in vivo CaMKII site if the anti-Thr(P)-787 antibody is used to directly stain transfected tsA201 cells. For these experiments, cells were transfected with GFP-tagged Eag cDNAs, with or without the constitutively active form of CaMKII. After fixation, cells were stained with the anti-Thr(P)-787 antibody. Confocal images are shown in Fig. 5. Images on the left show GFP fluorescence, which represents Eag expression level and localization in these cells. Images of the same field on the right side of Fig. 5 show rhodamine fluorescence, which represents anti-Thr(P)-787 staining. Eag appears to be on plasma membranes as well as in internal membrane compartments, consistent with the fact that Eag is a membrane protein. The pattern of Eag localization does not Purified GST-Eag-C2 and GST-Eag-C2-T787A fusion proteins were phosphorylated by CaMKII in reaction mixture with (ϩ) or without (Ϫ) calcium and calmodulin. Proteins were separated by SDS-PAGE and immunoblotted with affinity-purified anti-Thr(P)-787 antibody. Only phosphorylated GST-Eag-C2 (indicated by the arrow) and its degradation products are detected. B, detection of phosphorylation of Thr-787 in tsA201 cells. Cells were transfected with Myc-tagged Eag cDNA alone or with CaMKII-T287D. Eag was immunoprecipitated from cell extracts with anti-Eag N-terminal antibody. Immunoprecipitates were divided into two parts, separated by SDS-PAGE, and subjected to immunoblotting. The left half of the blot was probed using an anti-Myc monoclonal antibody to show that equal amounts of Eag were immunoprecipitated (IP) from each extract. The right half of the blot was probed with anti-Thr(P)-787 antibody to detect Eag phosphorylation. The arrow marked Eag indicates the position of full-length Eag. The lower molecular weight bands are rabbit immunoglobulin and are indicated by an arrow marked Ig. This experiment was performed three times with equivalent results. seem to be altered by the co-expression of CaMKII. When Eag-WT was expressed alone (top panels), some cells showed Thr-787 phosphorylation, which could be attributed to the endogenous CaMKII activity (a finding consistent with Fig. 4B). When GFP-Eag-WT was co-expressed with CaMKII (middle panels), almost all cells showed rhodamine fluorescence and the signal was stronger than that seen in the top right panel, indicating a higher level of phosphorylation of Eag at Thr-787. When the GFP-Eag-T787A mutant was co-expressed with CaMKII (bottom panels), there was GFP fluorescence but no rhodamine fluorescence. These results confirm that, in tsA201 cells, Eag is phosphorylated at Thr-787 by CaMKII. In addition, these experiments demonstrate that the anti-Thr(P)-787 antiserum gives highly specific cell staining.
Threonine 787 Is Phosphorylated in Vivo in Drosophila Neurons-To determine whether Thr-787 was phosphorylated by CaMKII in vivo in flies, we employed the Drosophila GAL4-UAS system to drive overexpression of CaMKII in neurons and probed Eag phosphorylation with the phosphospecific anti-Thr(P)-787 antibody. C155, a GAL4 line that drives expression in the nervous system, was crossed to UAS-CaMKII-T287D (16). Male third instar larvae of the progeny from this cross were dissected and their body wall muscles stained with the anti-Thr(P)-787 antibody. To look at basal Eag phosphorylation, we stained male larvae from a cross of C155-GAL4 to Canton S wild type flies. Male eag sc29 larvae (which are null for the full-length eag mRNA 13) and male larvae from a cross of UAS-CaMKII-T287D to C155-GAL4 containing the eag1mutant allele (which deletes the Thr-787 region) 2 were also examined. Fig. 6 shows images taken at identical settings of the confocal microscope of larvae processed in tandem. In all pictures, abdominal segment 3, muscles 6 and 7, are shown. Nerve terminals are indicated by arrowheads. Light staining is seen in control (C155-GAL4 X Canton S) animals. The most prominent staining at the nerve terminal is seen in larvae expressing constitutively active CaMKII. This suggests that Eag is expressed in the axon and terminal boutons of larval motor neurons and that phosphorylation is increased by expression of the CaMKII-T287D transgene. There is almost no neuronal signal observed in eag sc29 larvae stained with this antibody, which is not surprising, because this line does not make full-length eag mRNA. C155-GAL4 eag 1 larvae also show very little staining. These results suggest that anti-Thr(P)-787 antibody is specific for the phosphorylated Thr-787 in vivo in the nerve terminals. Staining in the nuclei of muscle cells is seen in both wild type and eag sc29 mutant larvae. This may be nonspecific or it may be caused by the presence of a 50-kDa fragment of the C terminus that is still produced in eag sc29 (data not shown). Overall, this result strongly suggests that Thr-787 of Eag is phosphorylated in vivo by CaMKII.
Phosphorylation of Thr-787 Modulates Eag Activity-To determine the functional consequences of Thr-787 phosphorylation, we expressed Eag in Xenopus oocytes. Because the level of activity of CaMKII endogenous to oocytes is unknown, we considered the possibility that endogenous CaMKII already might have phosphorylated the oocyte-expressed channels. If so, it should be possible to modulate Eag activity by inhibiting the endogenous CaMKII, providing there is an active phosphatase. Some of the most potent and specific inhibitors of CaMKII activity characterized to date are peptides with sequences corresponding to the autoinhibitory region of CaMKII (21). Fig. 7A (left) shows the current-voltage relations obtained for shamversus autoinhibitory peptide-injected oocytes expressing wild type Eag channels. In the presence of the peptide, Eag peak current amplitudes, measured in response to test pulses to ϩ40 mV (holding potential, Ϫ80 mV), decreased by nearly 50%, from 4.0 Ϯ 0.1 A to 2.1 Ϯ 0.1 A (n ϭ 8, p Ͻ 0.001; actual current values for the normalized data shown in Fig. 7A, left). Comparison of the current-voltage relations shows that the percent decrease in amplitude appeared to be uniform for all voltages within the activation range of the channel (Fig. 7A,  left). Peak amplitudes at ϩ40 mV also were decreased in the presence of another inhibitor of CaMKII (22), lavendustin C (18 M), from 4.1 Ϯ 0.2 A to 2.9 Ϯ 0.1 A (n ϭ 9 for both conditions, p Ͻ 0.001; data not shown).
The inhibitory effects of autoinhibitory peptide and lavendustin C were dependent on Thr-787, the residue shown to be phosphorylated by CaMKII in our in vitro phosphorylation assays. Fig. 7A (right) shows the average current-voltage relations obtained for sham-versus inhibitory peptide-injected oocytes expressing Eag-T787A channels. To allow direct comparison with the effect of the peptide on wild type channels, the current-voltage relations were normalized to the maximum peak current observed in control oocytes in each case. Inhibitory peptide produced no change in the amplitudes of Eag-T787A currents at any voltage (Fig. 7A, right). Similarly, there was no change in the average current-voltage relations following treatment with lavendustin C (data not shown). Peak amplitudes of Eag-T787A currents elicited by test pulses to ϩ40 mV were 1.1 Ϯ 0.1 A (n ϭ 9) and 1.0 Ϯ 0.1 A (n ϭ 7) for control and lavendustin C-treated oocytes, respectively.
Together, these results suggest that CaMKII endogenous to oocytes phosphorylates Eag channels and that phosphorylation at Thr-787 produces a substantial increase in Eag current. If, as the above results suggest, the CaMKII endogenous to oocytes is normally active in stage V and VI oocytes, one also would predict a difference in the current amplitudes of Eag-WT and Eag-T787A channels following injection of equal amounts of RNA. In agreement with this idea, Fig. 7B shows that the average peak current elicited by test pulses to ϩ40 mV was 70% smaller for Eag-T787A than for the wild type channel (p Ͻ 0.001). Substitution of aspartate or glutamate residues, which adds a single negative charge at the phosphorylation sites, has been shown in several instances to totally or partially mimic the effects of phosphorylation (23,24). As shown in Fig. 7B, injection of an equal amount of Eag-T787D RNA produced current amplitudes that were larger than the Eag-T787A amplitudes by 1.5-fold (p Ͻ 0.001 for comparison to T787A) but not equivalent to wild type amplitudes. Given that phosphorylation results in the addition of more than one negative charge to the side chain, one interpretation of this result is that wild type channels expressed in oocytes are maximally phosphorylated. The addition of phosphate to the threonine residue may have a larger effect on amplitude than the substitution of aspartate because of the increased amount of negative charge. This type of quantitative difference between a phosphorylated residue and an acidic substitution has been seen in other mutants (24). Finally, in addition to the above noted effects on amplitude, phosphorylation at Thr-787 also slows the inactivation of Eag channels. As shown in Fig. 7C and Table I, when compared with currents recorded from wild type channels, a larger proportion of the T787A current was inactivating and inactivation kinetics also were significantly faster. Similar trends also were observed in oocytes treated with inhibitory peptide and lavendustin C, although only the results for lavendustin C reached statistical significance (Table I). Alteration of Thr-787 did not appear to affect either the activation kinetics (Table I) or the voltage dependence of Eag channels (data not shown).

DISCUSSION
Previous studies from our laboratory (7) have shown that inhibition of CaMKII activity, by transgenic expression of a CaMKII autoinhibitory peptide (the ala transgene), results in electrophysiological and behavioral phenotypes that bear a striking similarity to those observed in Drosophila eag mutants. The simplest interpretation of these results is that CaMKII phosphorylation of Eag channels normally maintains or enhances Eag activity. In support of this hypothesis, the results of the present study demonstrate that CaMKII phosphorylates Eag at Thr-787 and that changes in phosphoryla-tion at this site modulate current amplitude. Mutations of Eag at the CaMKII phosphorylation site that prevent phosphorylation (T787A) or mimic phosphorylation (T787D) result in significant changes in current amplitude. These changes must be the result of either alterations in channel properties or alterations in channel processing. The additional effect on channel inactivation, which also is consistent with the larval electrophysiological phenotype, suggests that effects on channel properties are the more likely interpretation.
This study also provides a likely explanation for one aspect of the eag phenotype at the NMJ. The eag phenotype observed in recordings at the larval NMJ is particularly distinctive, exhibiting several characteristics that are not mimicked by mutations of other potassium channel genes, such as Shaker (Sh) or slowpoke (6,25). In particular, eag mutants have both spontaneous activity generated in the axon and an abnormal response to stimulation. Each stimulus is followed by multiple prolonged excitatory junctional potentials and an afterdischarge that continues for several minutes following the complete cessation of stimulation. These observations have suggested that one function of Eag channels is to limit excitability by maintaining the resting potential of presynaptic motor nerve terminals, although it has remained unclear whether the phenotype is a consequence of the loss of Eag channels directly from the terminal or whether, for example, the phenotype is a consequence of a more distant loss resulting in activity-dependent regulation of the expression of multiple other potassium channel types (26). Although not eliminating activity-dependent regulation as a possible contributor to the eag phenotype, our results provide the first demonstration of the localization of Eag directly at the presynaptic motor nerve terminals. In addition, we show that the level of Eag phosphorylation at this site is regulated by the activity of CaMKII. These data suggest that either the absence of Eag or the lack of CaMKII phosphoryla- tion of Eag at the terminal is likely to at least contribute to, if not account for, the supernumerary firing phenotype observed at the larval NMJ of eag mutants and autoinhibitory peptide (ala) transgenics.
The eag spontaneous activity phenotype is more difficult to assign to its interaction with CaMKII because it is not phenocopied in the ala transgenic. In fact, the spontaneous firing in eag is suppressed by ala when the transgene is expressed on an eag background (7) or when KN93 is applied to eag mutants. 3 This suggests that Eag may have regulators other than CaMKII and that CaMKII has ion channel targets other than Eag. Expression of constitutively active CaMKII has effects on neuronal excitability (27). Some of these effects appear to be mediated by up-regulation of potassium conductances. Our data suggest that Eag is one of the channels that is modulated by CaMKII, but it may not be only one.
The direct interaction of Eag and CaMKII also supports the idea that the memory formation phenotype of ala transgenics may be a result of changes in excitability. Mutations in Shaker, nap, and eag have been shown to disrupt courtship conditioning (28). The biochemical connection between Eag and CaMKII demonstrated in this study suggests that at least part of the reason that ala transgenics fail in this behavior may be defective modulation of Eag. It is probable that there are other 3 J. C. Choi, D. Park, and L. C. Griffith, unpublished results.

FIG. 7. CaMKII effects on Eag function.
A, comparison of current-voltage relations for sham-and inhibitory peptide-injected oocytes expressing Eag-WT (left) or Eag-T787A (right) channels. Oocytes were injected with 500 M amounts of the inhibitory peptide 15-90 min prior to recording. Currents were elicited by a series of voltage steps from Ϫ100 to 80 mV (holding potential, Ϫ80 mV). Wild type and T787A amplitudes were normalized to the maximum peak current observed in control conditions in each case. Currents were leak subtracted to reduce contamination of measurements by a leak current that was often induced by treatment with either the inhibitory peptide or lavendustin C, but not buffer alone. This current also was induced in oocytes not expressing Eag. Leak subtraction was performed using a P/4 pulse protocol with pulses of opposite polarity preceding each test pulse (holding potential, Ϫ80 mV). B, average peak current amplitudes observed in oocytes injected with equal amounts of RNA (0.16 ng/oocyte) encoding either wild type, T787A, or T787D channels. Recordings were made from oocytes obtained from the same frog on the same day following RNA injection. Oocytes were visually judged to be of the same stage and similar diameter. In addition, there was no appreciable difference in capacity measurements (obtained using 20-mV pulses from Ϫ80 mV) for the different conditions. C, scaled, superimposed traces obtained from Eag-WT and Eag-T787A expressing oocytes as indicated. In both B and C, currents were recorded on day 4 after RNA injection and were elicited by test pulses to ϩ40 mV (holding potential, Ϫ80 mV).

TABLE I Eag kinetics
Kinetic measurements were obtained from currents elicited by a test pulse to ϩ40 mV (holding potential ϭ Ϫ80 mV). The % inactivation was obtained using [(1 Ϫ I steady state /I peak ) ϫ 100], where I steady state was the mean current observed between 200 and 220 ms. This measurement could not be accurately determined (ND) for inhibitory peptide-injected oocytes due to contamination of the later portion of the traces by the appearance of a slow outward current. Measurements are the means Ϯ S.E. with the number of observations given in parentheses. The p values refer to t test comparisons with the control (NS ϭ not significant). Each grouping of data was obtained from recordings on the same day after RNA injection from oocytes of the same frog. excitability-related targets of CaMKII in addition to Eag, but the identity of these targets and the nature of CaMKII's interaction with them remain to be determined.