A Major Second Messenger Mediator of Electrophorus electricus Electric Tissue is CaM Kinase II

Comp. Biochem. Physiol. Vol. 118A, No. 1, pp. 81–91, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00411-2

A Major Second Messenger Mediator of Electrophorus electricus Electric Tissue is CaM Kinase II Anthony L. Gotter, Marcia A. Kaetzel, and John R. Dedman Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, U.S.A.

ABSTRACT. Electric tissue of the electric eel, Electrophorus electricus, has been used extensively as a model system for the study of excitable membrane biochemistry and electrophysiology. Membrane receptors, ion channels, and ATPases utilized by electrocytes are conserved in mammalian neurons and myocytes. In this study, we show that Ca 21 predominates as the major mediator of electric tissue phosphorylation relative to cyclic AMP and cyclic GMP-induced phosphorylation. Mastoparan, a calmodulin inhibitor peptide, and a peptide corresponding to the pseudosubstrate region of mammalian calmodulin-dependent protein kinase II (CaMKII (281–302)) attenuated Ca 21-dependent phosphorylation in a dose-dependent manner. These experiments demonstrated that calmodulin-dependent protein kinase II activity predominates in electric tissue. The Electrophorus kinase was purified by a novel affinity chromatography procedure utilizing Ca 21 /calmodulin-dependent binding to the CaMKII (281–302) peptide coupled to Sepharose. The purified 51 kDa calmodulin-dependent protein kinase II demonstrated extensive autophosphorylation and exhibited a 3- to 4-fold increase in Ca 21-independent activity following autophosphorylation. Immunofluorescent localization experiments demonstrated calmodulin to be abundant in electrocytes, particularly subjacent to the plasma membrane. Calmodulin-dependent protein kinase II had a punctate distribution indicating that it may be compartmentalized by association with vesicles or the cytoskeleton. As the primary mediator of phosphorylation within electric tissue, CaM kinase II may be critical for the regulation of the specialized electrophysiological function of electrocytes. comp biochem physiol 118A;1:81–91, 1997.  1997 Elsevier Science Inc. KEY WORDS. Electrophorus electricus, calmodulin-dependent protein kinase, membrane excitability, peptide inhibitors

INTRODUCTION The main electric organ of the electric eel, Electrophorus electricus, is the most powerful bioelectric generator in the animal kingdom (13), and represents an exaggerated model system in which to study the regulation of excitable membranes. Specialized cells of the electric organ produce membrane potentials that give rise to whole animal electrical discharges of up to 600 volts in the water surrounding the eel (20). Electrocytes develop embryonically from skeletal muscle, and retain morphological, biochemical and electrophysiological properties of progenitor-myocyte membranes. Membrane proteins involved in generating electrocyte potential changes are functionally identical to those of mammalian skeletal muscle. Because these membrane proteins exist in such high abundance in eel electric tissue, many investigators have used it as a source to examine the strucAddress reprint requests to: John R. Dedman, Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, P.O. Box 670576, Cincinnati, OH 45267-0576. Tel. (513) 558-4145; Fax (513) 558-5738. Current address of Anthony L. Gotter: Laboratory of Developmental Chronobiology, Harvard Medical School, Boston, MA. Received 16 August 1996; accepted 2 October 1996.

ture and pharmacology of the Na 1 channel, the nicotinic acetylcholine receptor, acetylcholinesterase, and the Na 1 / K 1 ATPase. Electrophorus electric tissue has also been found to be a rich source of the ubiquitous Ca 21 binding protein, calmodulin (4). In fact, roughly 2% of electric tissue protein is calmodulin (32). Hormones and neurotransmitters act through various second messenger signal transduction pathways to effect cellular processes. Second messengers such as cyclic nucleotides and Ca 21 activate serine/threonine kinases which affect cell physiology by phosphorylating membrane receptors and ion channels that control membrane excitability (23). Cyclic AMP-dependent protein kinase (PKA) and cyclic GMPdependent protein kinase (PKG) can phosphorylate multiple muscle membrane receptor and ion channel substrates. Phosphorylation by PKA, for example, potentiates the activity of Ca 21 channels in skeletal muscle (37,41), enhances desensitization of nicotinic acetylcholine receptors (17), and reduces peak Na 1 currents (9). Ca 21, another second messenger, activates protein kinase C (PKC) (2), as well as calmodulin-dependent protein kinases. PKC phosphorylation of nicotinic acetylcholine receptors results in prolonged desensitization of the receptor

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similar to that of PKA phosphorylation (10,8). PKC also decreases the conductance and prolongs the inactivation of skeletal muscle Na 1 channels (34,49). Ca 21 also activates kinases by interacting with calmodulin. Unlike other calmodulin-dependent protein kinases, calmodulin-dependent protein kinase II (CaM kinase II) has been found to have widespread tissue distribution and multisubstrate specificity (5,39). Initial studies have focused on the function of CaM kinase II in the mammalian central nervous system where its abundance is unusually high (11). Nevertheless, in smooth muscle and in cardiac muscle, CaM kinase II has been shown to modulate ion channels leading to changes in membrane excitability. For example, CaM kinase II is responsible for Ca 21-dependent enhancement of voltagedependent Ca 21 current in smooth muscle myocytes (28), as well as cardiac myocytes (1). However, the role of CaM kinase II in the modulation of skeletal muscle membrane excitability has not been well established. This investigation examines the second messenger pathways involved in the phosphorylation of electric tissue proteins. Regulation of electrocyte membrane excitability by second messenger pathways will lend insight into the function of kinases in regulating the electrophysiology of other excitable cells. MATERIALS AND METHODS Materials Electrophorus electricus, electric eels, were obtained from Worldwide Animals (Tampa, FL). ATP[γ 32P] was purchased from Dupont New England Nuclear at specific activities of 30 Ci/mmole for second messenger phosphorylation assays, and 3000 Ci/mmole for Ca 21-induced Ca 21-independent activity assays. Calmodulin was purified from rat testes as described previously (6). A synthetic peptide corresponding to amino acids 281–302 of the rat brain CaM kinase II αisoform (CaMKII (281–302)) was synthesized and analyzed by mass spectroscopy at the Analytical Chemistry Center at the University of Texas, Houston. Calmodulin and the CaMKII (281–302) peptide were coupled to CNBr activated Sepharose 4B (Pharmacia) according to the manufacturer’s instructions. Fluorescein-labeled secondary antibodies were purchased from Cappel. All other chemicals and reagents were obtained from Sigma Chemical Co. unless otherwise noted. Phosphorylation of Electric Tissue Proteins Live electric eels, under 3-aminobenzoic acid anesthesia, were removed from freshwater aquariums and 4 mm biopsy plugs (0.1 g) were extracted from the main electric organ. After the skin was removed by dissection, electric tissue was homogenized in 50 volumes of 50 mM HEPES buffer (pH 7.4), 0.15 M NaCl, 10 mM Mg acetate, 7 mM 2-mercaptoethanol, 2 mM EGTA, and 200 µM phenylmethylsulfonyl

fluoride (PMSF) at 4°C. Nuclei and mitochondria were separated by centrifugation at 2,500 g for 10 min. For second messenger-dependent phosphorylation assays, electric tissue extracts were brought to 1.2 mM CaCl 2 (0.2 mM Ca 21 excess), cyclic AMP (10 µM and 100 µM), cyclic GMP (10 µM and 100 µM), PMA (10 nM and 1 µM) or 1 µg/ml bovine heart PKA catalytic subunit in a total volume of 50 µl. Reactions were preincubated at 30°C for 90 sec to insure that the tube and the reaction mixture were maintained at the proper temperature. Assays were initiated by adding 10 µM ATP [γ 32P] (30 Ci/mmole) and terminated after 20 sec by 20% trichloroacetic acid (TCA) precipitation. Proteins were pelleted by centrifugation at 10,000 g for 10 min, and solubilized in sample buffer prior to analysis by SDS-PAGE and autoradiography. Assay duration was determined empirically by assessing the time for peak phosphorylation to occur. With a 1 : 50 dilution of electric tissue in reaction buffer at 30°C, peak phosphorylation of electric tissue proteins occurred at 30 sec. In order to assess calmodulin and CaM kinase II-dependent phosphorylation of electric tissue proteins, mastoparan (1 µM and 10 µM), a peptide inhibitor of calmodulin, or the CaMKII (281–302) peptide (10 µM and 20 µM), a specific inhibitor of CaM kinase II (25,46), were added to reaction mixtures prior to preincubation. To assess collective phosphatase activity within electric tissue extracts, 100 µl reaction mixtures were prepared and phosphorylation of endogenous proteins was carried out in the presence of 10 µM ATP [γ 32P] (30 Ci/mmole) for 30 sec, as described above. Unlabeled ATP to 10 mM was added to the reaction to dilute ATP[γ 32P] 1000-fold to terminate radiolabeled phosphate incorporation, and to simultaneously assess dephosphorylation. At indicated times, 25 µl aliquots were taken from the reaction and immediately placed in an equal volume of 20% TCA to stop the reaction. Each aliquot was subjected to SDS-PAGE followed by autoradiography. Ca 21-induced Ca 21-independent Activity of Electrophorus CaM Kinase II Fifty µl reactions were prepared as described above to determine Ca 21-induced Ca 21-independent activity of electric tissue CaM kinase II following autophosphorylation. These assays measured the ability of an Electrophorus kinase to phosphorylate autocamtide-2 (Peninsula Labs), a peptide substrate specific for CaM kinase II. The K m of CaM kinase II for this substrate is approximately 20 µM, a concentration of autocamtide-2 that shows little phosphorylation by other known kinases (15). Reactions were carried out in the following solution held at 4°C prior to incubation: 50 mM HEPES buffer (pH 7.4), 0.15 M NaCl, 10 mM Mg acetate, 7 mM 2-mercaptoethanol, 2 mM EGTA and 200 µM PMSF, 50 µM ATP and 20 µg/ml calmodulin. Assays were prepared by preincubating the mixture for 90 sec at 30°C followed by a 45-sec autophosphorylation period. The sub-

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strate phosphorylation reaction was then initiated with the addition of ATP[γ 32P] to 1.0 Ci/mmole along with autocamtide-2 to 20 µM, and terminated by adding ice cold EDTA to 50 mM. Reaction aliquots were then spotted onto phosphocellulose filters (Whatman) for scintillation counting as previously described (36). For Ca 21-dependent phosphorylation assays, 2.2 mM CaCl 2 (0.2 mM Ca 21 excess) was added just prior to the preincubation period and was present throughout the reaction. To measure Ca 21-independent activity, Ca 21 was absent throughout the entire procedure (2 mM EGTA excess). For assays measuring Ca 21-independent activity following autophosphorylation, Ca 21 was added to initiate the 45-sec autophosphorylation period. Autophosphorylation was then terminated by bringing the mixture to 10 mM EGTA just before initiation of the phosphorylation reaction with ATP[γ 32P] and autocamtide2. Ca 21-independent and Ca 21-induced Ca 21-independent activity following autophosphorylation were expressed as a percent of Ca 21-dependent activity. Purification of Electrophorus CaM Kinase II Soluble main electric organ proteins were prepared as previously described (18). The soluble fraction was then dialyzed against 10 mM imidazole (pH 7.4), 1 mM EGTA and 175 mM NaCl (buffer A). Endogenous calmodulin was removed from the dialysate by applying this fraction to DEAE ionexchange resin (Whatman) previously equilibrated with buffer A. After the resin was washed with 5 volumes of buffer A, unbound and wash fractions, devoid of calmodulin, were pooled. Calmodulin Sepharose affinity chromatography was then performed as previously described (18). Fractions containing calmodulin binding proteins were eluted in 50 mM HEPES buffer (pH 7.4), 0.15 M NaCl, 10 mM Mg acetate, 7 mM 2-mercaptoethanol, 2 mM EGTA (buffer B) and pooled. To purify CaM kinase II from other calmodulin binding proteins, affinity chromatography utilizing the CaMKII (281–302) peptide coupled to Sepharose was performed, the mechanism of which is depicted in Fig. 4. Pooled fractions containing calmodulin binding proteins were brought to 2.2 mM CaCl2 and 100 µg/ml calmodulin and loaded onto a 1 ml CaMKII (281–302)-Sepharose column (2.5 mg peptide/ml resin), which had been equilibrated with buffer B plus 2.2 mM CaCl2. After washing the column with 5 volumes of buffer B plus 2.2 mM CaCl2, elution was accomplished with buffer B containing no added Ca 21 (2.0 mM EGTA excess). Fractions (0.5 ml) were collected and protein content estimated by dot blot/Ponceau S stain analysis. The ability of CaMKII (281–302) peptideSepharose to bind CaM kinase II decreased rapidly with age of the peptide and use of the affinity resin. Nevertheless, freshly coupled CaMKII (281–302) peptide-Sepharose was routinely used to purify CaM kinase II from other calmodulin binding proteins. The presence of CaM kinase II in each fraction during purification was determined by phosphoryla-

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tion assays followed by SDS-PAGE and autoradiography as described above. Immunoblot analysis was performed as previously described (18, 19), and probed with monospecific affinity-purified sheep antibody against annexin VI or sitespecific rabbit antibody directed against the amino-terminus of the α-isoform of rat CaM kinase II. RESULTS Second Messenger Induced Phosphorylation of Endogenous Substrates Signal transduction pathways rely on second messengers to activate protein kinases, which in turn modulate cell function through phosphorylation. The relative activities of endogenous Ca 21 and cyclic nucleotide-dependent kinases toward endogenous electric tissue substrates were evaluated with phosphorylation assays in which electric organ extracts were incubated with ATP[γ 32P]. Extracts were then subjected to SDS-PAGE and phosphoproteins visualized by autoradiography. Elevated free Ca 21 (0.2 mM) dramatically increased the phosphorylation of numerous electric tissue proteins (Fig. 1A). Cyclic AMP, however, at concentrations of 10 and 100 µM induced little phosphate incorporation, indicating that PKA activity is imperceptible relative to Ca 21-dependent activity. When exogenous PKA catalytic subunit was added, it phosphorylated numerous proteins. This indicated that if PKA were present in appreciable endogenous concentrations, it would have mediated phosphorylation during the 20-sec assay. When cyclic GMP was included in the reaction, no phosphate incorporation into endogenous substrates was seen, which demonstrates that little steady-state PKG activity exists in this tissue relative to Ca 21-dependent activity. Most isoforms of PKC are dependent upon Ca 21 and diacylglycerol for their activation (2), and may have been responsible for the Ca 21-dependent phosphorylation seen in these assays. In order to further investigate the nature of the Ca 21-dependent phosphorylation, PMA was used as a potent Ca 21-independent activator of PKC (44). PMA at concentrations of 10 nM and 1 µM, however, triggered no detectable protein phosphorylation, indicating that the Ca 21-dependent phosphorylation was not due to PKC. Identification of the Ca 21-dependent Kinase Activity Calcium can also mediate phosphorylation through the activation of calmodulin-dependent protein kinases. Calmodulin-activated enzymes have regulatory domains that include both a pseudosubstrate region as well as a calmodulin binding site. In the inactive state, the pseudosubstrate domain occupies the active site and the protein is inhibited. Calmodulin activates these enzymes in the presence of Ca 21 by binding to the regulatory domain at a site adjacent to the pseudosubstrate region, reversing the autoinhibition of the regulatory domain. In order to determine the role of

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FIG. 1. Phosphorylation of electric tissue proteins by endogenous protein kinases. A. Second messenger-mediated phosphoryla-

tion of electric tissue proteins. Electric organ extracts in reaction buffer were incubated in the presence of ATP[g 32P] at 30°C for 20 sec before termination with TCA. Electric tissue proteins were subjected to SDS PAGE and those proteins incorporating 32 PO4 were visualized by autoradiography. In the indicated lanes, 0.2 mM Ca21, or specified concentrations of cyclic AMP, cyclic GMP, exogenous PKA catalytic subunit, or PMA were included in the reaction mixtures. Migration of molecular weight standards are indicated to the left of the autoradiogram. B. Calmodulin and CaM kinase II dependent phosphorylation of electric tissue proteins. Electric organ extracts were incubated with ATP[g 32P] at 30°C for 20 sec in the absence or presence of 0.2 mM Ca 21 and the indicated concentrations of mastoparan or the CaMKII (281–302) peptide.

calmodulin in Ca 21-dependent phosphorylation of electric tissue proteins, mastoparan, a wasp venom peptide toxin, was used to potently inhibit calmodulin. When mastoparan is included in phosphorylation reactions, Ca 21-dependent phosphate incorporation is attenuated in a dose dependent fashion (Fig. 1B). This indicates that Ca 21-dependent phosphorylation is mediated by calmodulin. The major band that incorporated 32PO4 in a Ca 21-dependent manner migrates at approximately 51 kDa, a molecular weight similar to the α-isoform of rat brain CaM kinase II. The mammalian kinase is also known to extensively autophosphorylate and have multisubstrate specificity (15,16,30). Peptide inhibitors of CaM kinase II have been utilized in numerous studies to probe the role of the kinase in physiological function. These peptides correspond to the pseudosubstrate region and specifically inhibit CaM kinase II without appreciably binding calmodulin (45,46). This region has .98% sequence homology among known rat isoforms as well as the α-isoform of human CaM kinase II (3,24,33). Inhibition of kinase activity by peptides with this sequence

is therefore indicative of the presence of CaM kinase II. To investigate the extent of CaM kinase II-dependent phosphorylation of electric tissue proteins, we used a peptide corresponding to amino acids 281–302 of the α isoform of rat brain CaM kinase II (CaMKII (281–302)), which has been used previously to elucidate the role of the kinase in cell function (25). When included in phosphorylation reactions, this peptide attenuated Ca 21-dependent phosphate incorporation into electric organ proteins (Fig. 1B). These data indicated that a teleost form of CaM kinase II was inhibited by a regulatory domain peptide whose sequence is conserved among many species. Taken together, the data of Fig. 1 suggest that CaM kinase II-dependent phosphorylation predominates in electric tissue, and is a major second messenger mediator. Dephosphorylation of Endogenous Substrates Steady-state phosphorylation is determined by a delicate balance between protein kinases and opposing phosphatase

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more delayed (Fig. 2, ‘‘Unlabeled ATP 1 EGTA’’). These data indicate that collective phosphatase activity is much slower compared to Ca 21-dependent kinase activity. Under stimulated conditions where intracellular Ca 21 levels are increased within electric tissue, CaM kinase II activity dominates and shifts the steady-state balance of phosphorylation. Affinity Purification of Electrophorus CaM Kinase II

FIG. 2. Ca21-dependent and Ca 21-independent dephosphor-

ylation of electric tissue phosphoproteins. Electric Tissue proteins were phosphorylated for 30 sec at 30°C in the presence of 0.2 mM free Ca 21, as described in Fig. 1, except that 100 ml reaction volumes were prepared (lane 1). Dephosphorylation activity was assessed by adding unlabeled ATP to 10 mM in order to terminate 32PO4 incorporation. At the indicated times, 25 ml aliquots were immediately transferred to eppendorf tubes containing 20% TCA to terminate activity. Precipitated proteins were solubilized in sample buffer and analyzed by SDS-PAGE and autoradiography (‘‘Unlabeled ATP only’’). For Ca 21-independent dephosphorylating activity, unlabeled ATP and EGTA were added simultaneously to 10 mM concentrations (‘‘Unlabeled ATP 1 EGTA’’). As a negative control, 10 mM unlabeled ATP was included in the 30-sec phosphorylation reaction before the addition of ATP[g 32P].

activities. Dephosphorylation assays were performed to evaluate collective electric organ phosphatase activities in order to be compared to the rapid and extensive phosphorylation that occurs in the presence of Ca 21. Dephosphorylation assays were done by first incubating extracts with ATP[γ 32P] to incorporate phosphate into electric tissue proteins. An excess of unlabeled ATP was then added to the reaction in order to dilute ATP[γ 32P] 1000-fold, effectively terminating 32PO4 incorporation by endogenous kinases. Radiolabeled phosphate was then removed from phosphoproteins at a rate proportional to general phosphatase activity within the extract. In contrast to Ca 21-dependent phosphorylation, which peaks after 30 sec, dephosphorylation is relatively slow. In the presence of Ca 21, radiolabeled phosphoprotein substrates are detected even after 150 sec. Approximately 10% of the radiolabel remains incorporated on the 51 kDa kinase (Fig. 2, ‘‘Unlabeled ATP’’). In the absence of Ca 21, the release of radiolabeled phosphate is even

The prominent 51 kDa phosphoprotein band seen in the autoradiograms discussed above was likely to be the Electrophorus form of CaM kinase II. This major electric tissue kinase activity was purified to examine its properties more closely to identify it as a homologous isoform of the mammalian protein. The activity of the kinase at each step of the purification was monitored by phosphorylation assays as performed earlier. Since autophosphorylated CaM kinase II appears as a salient 51 kDa band on autoradiograms of tissue extracts, this method proved to be a sensitive assay for the detection of the kinase. Purification was initiated with homogenization of main electric organ tissue followed by differential centrifugation yielding a fraction containing soluble proteins devoid of membranes. Endogenous calmodulin was removed by ion exchange chromatography. The calmodulin-free fraction was then subjected to Ca 21-dependent calmodulin-Sepharose affinity chromatography. The final step of the purification involved affinity chromatography utilizing a peptide-Sepharose resin (Fig. 3A). The peptide used for this affinity matrix is CaMKII (281– 302), the same peptide used previously to inhibit CaM kinase II activity in phosphorylation assays. This peptide interacts with both the ATP and peptide substrate binding sites of the kinase and competes with the endogenous regulatory domain of the kinase (12,46). When calmodulin binds to the kinase, the endogenous autoinhibitory region loses its affinity for the active site and the CaMKII (281– 302) peptide is able to bind the kinase (Fig. 3A, top). Before applying the calmodulin-binding protein fraction to the affinity matrix, Ca 21 and calmodulin were added in order to displace the endogenous autoinhibitory domain from the active site. Numerous calmodulin-binding proteins (Fig. 3B) were applied to CaMKII (281–302) peptide-Sepharose including CaM kinase II which autophosphorylated in Ca 21-dependent manner (panel C). After washing the column with Ca 21 containing buffer, the kinase was eluted with EGTA. Without Ca 21 /calmodulin, the endogenous autoinhibitory domain displaces the CaMKII (281–302) peptide and CaM kinase II and calmodulin are eluted (Fig. 3A, bottom). CaM kinase II, calmodulin and a 67 kDa protein eluted from CaMKII (281–302) peptide-Sepharose during the EGTA elution (Fig. 3B and C), demonstrating that these proteins adhered to the affinity resin in a Ca 21dependent manner. Immunoblot analysis identified the 67 kDa protein as annexin VI, a Ca 21-dependent phospholipid binding protein found in skeletal muscle (7,19). This pro-

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tein was selectively removed from the applied fraction and concentrated in the EGTA elution (Fig. 3C). In vitro, annexin VI did not alter autophosphorylation of the kinase, nor the activity toward autocamtide-2, a specific substrate of CaM kinase II. Purified annexin VI did bind to calmodulinSepharose, but did adhere to CaMKII (281–302) peptideSepharose in a Ca 21-dependent manner, suggesting that its copurification is not necessarily due to a direct association with CaM kinase II (not shown). Isoform Comparison of Mammalian and Electric Eel CaM Kinase II

FIG. 3. Isolation of CaM kinase II using CaMKII (281–302)

peptide-Sepharose affinity chromatography. A. Model for the purification of CaM kinase II by affinity chromatography utilizing Sepharose coupled to the CaMKII (281–302) peptide. Proteins are applied to the peptide Sepharose resin in the presence of added Ca21 (0.2 mM) and calmodulin (100 mg/ml). Ca 21 /calmodulin binds the kinase and makes the active site available for binding to the peptide-Sepharose. In the absence of Ca 21, calmodulin dissociates from the kinase, endogenous autoinhibitory domain replaces the peptide and the kinase elutes from the affinity resin. B. Coomassie Blue– stained SDS-PAGE gel of samples taken before (‘‘applied’’) and after (‘‘unbound,’’ ‘‘EGTA elution’’) affinity chromatography. Samples were solubilized in SDS PAGE sample buffer and loaded onto a 10% SDS polyacrylamide gel. Proteins not adhering to CaMKII (281–302)-Sepharose in the presence of Ca 21 appear in the unbound fraction. After washing the column with buffer B containing 150 mM NaCl and 0.2 mM Ca21, elution was performed with buffer B containing no added Ca 21 (2 mM EGTA excess). Calmodulin is absent from the ‘‘Applied’’ fraction as this sample was taken prior to the addition of the protein. C. Autophosphorylation of purified CaM kinase II. Phosphorylation of affinity chromatography fractions were performed as in Fig. 1. Samples of applied, unbound and EGTA eluted fractions were brought to 100 mg/ml calmodulin and 2 mM EGTA or 0.2 mM Ca 21 prior to initiating reactions with ATP[g 32P]. D. Immunoblot analysis of affinity chromatography fractions for the presence of annexin VI probed with monospecific polyclonal antibody against annexin VI.

Numerous isoforms of mammalian CaM kinase II have been identified and are distributed differentially in mammalian tissues. Isoforms specific to neuronal tissue have molecular weights of 50, 60, and 59 kDa and have been designated as the α, β, and β ′ subunits, respectively. Other isoforms having molecular weights of 59 kDa (γ ) and 60 kDa (δ ), have also been described in non-neuronal tissue including skeletal muscle (5,39,48). Figure 4 shows an immunoblot probed with an affinity-purified antibody raised against a peptide corresponding to the N-terminal 20 amino acids of the αisoform of rat brain CaM kinase II, and directly compares the eel kinase to rabbit brain CaM kinase II. This antibody defined the mobility of the rabbit brain kinase, particularly the 50 kDa α-subunit. It also recognized purified Electrophorus CaM kinase II, which migrates at a molecular weight slightly higher than the α-subunit of the rabbit brain kinase (Fig. 4). In accordance with autoradiograms that showed a single 51 kDa autophosphorylating protein, immunoblot analysis also demonstrated the presence of only one Electrophorus CaM kinase II isoform that migrates at a molecular weight similar to the α-isoform of the mammalian protein. Ca 21-induced Ca 21-independent Activity of Electrophorus CaM Kinase II A distinguishing characteristic of CaM kinase II is its ability to attain Ca 21-independent activity following autophosphorylation. The mechanism of this ‘‘Ca 21-induced Ca 21independent activity’’ involves autophosphorylation of a threonine residue on the autoinhibitory domain (16,30,40). After calmodulin activates the kinase, this threonine residue is made available for phosphorylation by an adjacent subunit of the kinase in the protein complex. As Ca 21 levels fall and calmodulin dissociates from the kinase, the autophosphorylated regulatory domain no longer inhibits the kinase due to steric hindrance of the phosphate moiety and an alteration if its secondary structure (16,31,40). The result is a kinase with Ca 21 /calmodulin-independent activity. Phosphorylation of autocamtide-2 by Electrophorus CaM kinase II was measured in the presence and the absence of Ca 21 to determine maximal and basal activity of the kinase, respectively (Fig. 5A). To measure Ca 21-independent activity

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Ca 21-dependent activity (Fig. 5B). After a period of Ca 21dependent autophosphorylation, however, the average activity in the absence of Ca 21 rose to 40.4 6 5.6% (p , 0.01), an increase of 3.8 6 0.7 fold. Like the mammalian kinase, Electrophorus CaM kinase II also exhibits Ca 21-induced Ca 21-independent activity. Immunolocalization of Calmodulin and CaM Kinase II in Electric Tissue

FIG. 4. Immunoblot analysis of rabbit brain CaM kinase II and Electrophorus CaM kinase II probed with antibody against the N-terminal 20 amino acids of the a-isoform of rat brain CaM kinase II. Proteins subjected to SDS-PAGE were transferred to nitrocellulose, probed with affinity purified anti-CaM kinase II antibody and visualized with horseradish peroxidase conjugated secondary antibody.

following autophosphorylation, reaction mixtures were incubated with Ca 21 and non-radioactive ATP to allow the kinase to autophosphorylate. After this period of Ca 21-dependent autophosphorylation, excess EGTA was added to the reaction just prior to the activity assay in order to measure Ca 21-independent activity. Basal activity of eel CaM kinase II in the absence of Ca 21 was 12.7 6 2.7% of the

Electric organ electrocytes have evolved a unique morphology endowing them the ability to produce transcellular potentials that summate to give rise to whole animal electrical discharges. These large ribbon-like cells are stacked regularly one after another along the length of the electric organ. Individual electrocytes are polarized such that acetylcholine receptors and voltage-dependent Na 1 channels lie in the electrically excitable innervated membrane, while the Na 1 /K 1-ATPase and resting current channels exist in the more undulated, non-innervated electrically inexcitable membrane. This polarization allows electrocytes to develop an asymmetric current flow during stimulation that is manifested as a transcellular potential, causing the electrocyte to act as a battery. In order to gain insight into the function of calmodulin and CaM kinase II in electric tissue, immunofluorescent localization of these proteins was performed. Electric tissue expresses calmodulin in extremely high levels. In fact, the concentration of the protein is greater in electrocytes than in any other tissue of Electrophorus (32). Affinity purified monospecific anti-calmodulin antibodies intensely stain electrocytes relative to surrounding neurons and blood vessels. Within electrocytes, calmodulin appeared to be concentrated along both the innervated and non-innervated membranes, while still being diffusely distributed throughout the cytoplasm (Fig. 6A). CaM kinase II was also highly concentrated within electrocytes (Fig. 6B), but with a distinctly punctate pattern of localization. Excluding nuclei, it localized to discrete points throughout the interior of the cell, suggesting that CaM kinase II is either compartmentalized or associated with intracellular structures. DISCUSSION Signal transduction pathways employ protein kinases to modulate cell function. CaM kinase II predominates over other modes of second-messenger mediated phosphorylation in Electrophorus electric tissue. This suggests that CaM kinase II is a major second messenger mediator regulating the specialized electrophysiological function of electrocytes. Other kinases may mediate phosphorylation of less abundant, yet critical substrates. However, to modulate the activity of exaggerated amounts of membrane proteins in electric tissue, rapid and extensive phosphorylation like that seen in the presence of Ca 21 would occur. Phosphorylation

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FIG. 5. Ca21-induced Ca 21-independent activity of Electrophorus CaM kinase II. A. Schematic time-course of experiments measuring Ca 21-independent activity of electric tissue extracts following Ca 21dependent autophosphorylation. Ca 21-dependent phosphorylation of autocamtide-2 was measured after a 45-sec autophosphorylation period. For basal Ca 21-independent activity, Ca 21 was excluded throughout the entire procedure. Ca 21-independent activity following autophosphorylation was measured after an autophosphorylation period that was initiated with Ca 21 and terminated with the addition of EGTA followed by the activity assay initiated with ATP[g32P] and substrate. B. Basal and autophosphorylation-induced Ca 21-independent activities are expressed as a percentage of autocamtide-2 phosphorylation in the presence of Ca 21. Data shown is one representative experiment with five trials (n 5 5). Each trial included assays for Ca21 dependent activity, basal Ca21 independent activity and Ca21-induced Ca 21-independent activity following autophosphorylation, as described in A.

dominates dephosphorylation under conditions of high Ca 21 when numerous proteins incorporate phosphate (Figs 1 and 2), suggesting that CaM kinase II shifts the balance of phosphorylation when electric tissue is stimulated. In order to identify Electrophorus CaM kinase II as a homologous isoform of the mammalian kinase, it was purified by exploiting its properties of selectively binding calmodulin and the CaMKII (281–302) peptide. The Electrophorus kinase readily autophosphorylates in a Ca 21 and calmodulin-dependent manner, similar to CaM kinase II from mammalian brain (16,30,40). This property was used in phosphorylation assays followed by autoradiography to detect the kinase in purification fractions. Only one isoform of the Electrophorus kinase was identified and purified. It migrated on SDS-PAGE at 51 kDa, a molecular weight similar to the α-isoform of the rabbit brain kinase, and reacted with antibody generated against a peptide corresponding to the N-terminus of the rat α-isoform. The Ca 21-independent ac-

tivity of the eel kinase increased nearly four-fold following autophosphorylation. The basal Ca 21-independent activity of 12.7% is high compared to basal activity of CaM kinase II from other tissues. Samples of electric organ were extracted while eels were actively discharging, so this elevated basal Ca 21-independent activity may actually represent kinase in a partially activated state. A Ca 21 /calmodulin-dependent kinase activity has also been partially purified and characterized from electric tissue of Torpedo californica, a marine electric ray (35). Analysis of exogenous substrate specificity indicated that this enzyme was CaM kinase II. Four major endogenous substrates were described, two of which having molecular weights of 54 and 62 kDa were presumably the kinase itself. Most of the Torpedo kinase activity was associated with presynaptic vesicles. This contrasts with Electrophorus electric tissue which expresses a single 51 kDa isoform that is localized within electrocytes. Electrophorus is of the teleostei order, whereas Tor-

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FIG. 6. Immunofluorescent localization of calmodulin (A) and CaM kinase II (B) within electric tissue electrocytes. Electrocytes are seen in cross-section where the flat innervated membrane lies to the left and the noninnervated more undulated surface is to the right. Regions between the cells are occupied by connective tissue, blood vessels, and efferent motor neurons. Main electric organ tissue was fixed overnight in 4% paraformaldehyde in PBS prior to embedding in paraffin. Four mm sections were then taken and mounted on slides. Nonspecific binding was blocked by applying 10% preimmune serum in PBS corresponding to the species of secondary antibody. Sections were probed with affinity-purified monospecific sheep antibody against calmodulin or affinity-purified rabbit antibody against the N-terminus of the a-isoform of rat CaM kinase II. Primary antibodies were visualized by applying fluorescein-labeled secondary antibody and viewing under epifluorescence microscopy. Control sections were prepared identically except that PBS replaced the primary antibody application. Micrographs of control sections were exposed for times identical to those sections stained for calmodulin and CaM kinase II. No appreciable fluorescence was seen in controls relative to experimental micrographs. Bar 5 25 mm.

pedo is an elasmobranch, and the existence of electric tissue in these two animals represents incomplete convergent evolution (14). Electrocytes of these two fishes are distinct biochemically, morphologically, and in their mechanism of membrane potential generation, and comparisons should be made with caution. Electrophorus annexin VI copurified with CaM kinase II. Purified annexin VI did not bind directly to calmodulinSepharose, and does not alter the activity of the kinase in vitro. However, purified annexin VI did bind to CaMKII (281–302)-Sepharose in a Ca 21 dependent manner. Annexin VI also occasionally copurified in amounts exceeding that of the kinase. This indicates that annexin VI and CaM kinase II do not directly interact during purification. Annexin VI was localized near the non-innervated membrane in immunofluorescent localization studies, a pattern distinct from both calmodulin and CaM kinase II (not shown). Immunofluorescent localization experiments identified calmodulin and CaM kinase II within electrocytes of the main electric organ. Calmodulin appeared concentrated near both innervated and noninnervated membranes. Since these cells are specialized to produce membrane potentials, calmodulin may play a role in the modulation of membrane receptors, ion channels, and ATPases. CaM kinase II displayed a punctate subcellular distribution within electrocytes. Electron microscopy revealed that the electrocyte cytoplasm is made up of a loose filamentous framework with scattered glycogen granules, but devoid of cellular organelles (27). CaM kinase II may be associating with these

structures. In rat hippocampal tissue, CaM kinase II translocates from the soluble fraction to the pellet fraction in response to ischemia (22), suggesting that the kinase is capable of associating with insoluble cellular structures such as vesicles or cytoskeletal proteins in response to intracellular signals. CaM kinase II has also been found to loosely associate with the membrane-cytoskeletal complex in Aplysia neurons, and is released upon autophosphorylation (38). In the present study, eel CaM kinase II has been purified from the soluble fraction, so its association with subcellular structures appears to be reversible. The eel kinase may also translocate in response to intracellular signals. Since calmodulin is necessary for initial activation of the enzyme, only those kinase molecules near the cell surface may be maximally active. CaM kinase II, therefore, may also have functions related to regulating changes in membrane potential. Electrically excitable cells respond to membrane depolarization with transient increases in intracellular calcium concentration. Ca 21-dependent kinases, like CaM kinase II, are poised to be activated by transient changes in calcium to catalyze the phosphorylation of cellular substrates. In fact, in ventricular myocytes CaM kinase II autophosphorylates in response to membrane depolarization and calcium entry (50). Numerous receptors and ion channels have been found to be phosphorylated by CaM kinase II, thereby regulating membrane excitability and cell function. In hippocampal neurons, where the α-isoform of the kinase is highly abundant (11), phosphorylation of glutamate receptors of the N-methyl-D-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) sub-

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types results in potentiated activity (21,29,47,51). The influence of CaM kinase II on glutamate receptors is believed to underlie behavioral memory. In fact, genetically altered mice with a disrupted α-CaM kinase II gene are deficient in spatial memory tasks (42,43). CaM kinase II has also been shown to augment the conductance of T-type Ca 21 channels in adrenal glomerulosa cells (26), and L-type Ca 21 channels of smooth muscle (28) and ventricular myocytes (1). Initial Ca 21 entry through these channels activates CaM kinase II activity and further augments Ca 21 conductance, potentiating subsequent responses. In cardiac ventricular myocytes, an increase in heart rate frequency may lead to increased CaM kinase II activation, a further increase in Ca 21 conductance and augmented myocardial contractility. Because calmodulin and CaM kinase II activity are so prevalent in electric tissue of the electric eel, they are likely to regulate the specialized electrophysiological function of electrocytes. Further study of the role of these proteins in regulating the electrophysiology of these cells is underway. Electrocytes perform their highly specialized function using membrane proteins common to other excitable cells and therefore provide an appropriate model system. Determining the role of calmodulin and CaM kinase II in membrane excitability of electric tissue will provide insight into the mechanisms of the regulation of ion channels, receptors, and ATPases in other excitable cells, particularly skeletal muscle. This work was supported by NIH grants DK41740 (MAK), DK46433 (JRD) and training grant HL07571 (ALG). The authors are grateful to Dr. Jorge M. Naciff for his insightful discussions.

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