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<!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.0 20120330//EN" "JATS-archivearticle1.dtd">
<article xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" article-type="research-article"><?properties open_access?><front><journal-meta><journal-id journal-id-type="nlm-ta">J Cell Biol</journal-id><journal-id journal-id-type="iso-abbrev">J. Cell Biol</journal-id><journal-id journal-id-type="publisher-id">JCB</journal-id><journal-title-group><journal-title>The Journal of Cell Biology</journal-title></journal-title-group><issn pub-type="ppub">0021-9525</issn><issn pub-type="epub">1540-8140</issn><publisher><publisher-name>The Rockefeller University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">16275756</article-id><article-id pub-id-type="pmc">PMC1343528</article-id><article-id pub-id-type="publisher-id">200505155</article-id><article-id pub-id-type="doi">10.1083/jcb.200505155</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Articles</subject><subj-group><subject>Article</subject></subj-group></subj-group></article-categories><title-group><article-title>CaMKII tethers to L-type Ca<sup>2<bold>+</bold></sup> channels, establishing a local and dedicated integrator of Ca<sup>2<bold>+</bold></sup> signals for facilitation</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Hudmon</surname><given-names>Andy</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Schulman</surname><given-names>Howard</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>James</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Maltez</surname><given-names>Janet M.</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Tsien</surname><given-names>Richard W.</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Pitt</surname><given-names>Geoffrey S.</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="aff" rid="aff5">5</xref></contrib></contrib-group><aff id="aff1">
<label>1</label>Department of Neurobiology, Stanford University, Stanford, CA 94305</aff><aff id="aff2">
<label>2</label>Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305</aff><aff id="aff3">
<label>3</label>Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, NY 10032</aff><aff id="aff4">
<label>4</label>Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, NY 10032</aff><aff id="aff5">
<label>5</label>Center for Molecular Cardiology, College of Physicians and Surgeons of Columbia University, New York, NY 10032</aff><author-notes><fn><p>Correspondence to Richard W. Tsien: <email>rwtsien@stanford.edu</email>
</p></fn></author-notes><pub-date pub-type="ppub"><day>7</day><month>11</month><year>2005</year></pub-date><volume>171</volume><issue>3</issue><fpage>537</fpage><lpage>547</lpage><history><date date-type="received"><day>24</day><month>5</month><year>2005</year></date><date date-type="accepted"><day>3</day><month>10</month><year>2005</year></date></history><permissions><copyright-statement>Copyright © 2005, The Rockefeller University Press</copyright-statement><license license-type="openaccess"><license-p>This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see <ext-link ext-link-type="uri" xlink:href="http://www.rupress.org/terms">http://www.rupress.org/terms</ext-link>). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-sa/4.0/">http://creativecommons.org/licenses/by-nc-sa/4.0/</ext-link>).</license-p></license></permissions><abstract><p>Ca<sup>2<bold>+</bold></sup>-dependent facilitation (CDF) of voltage-gated calcium current is a powerful mechanism for up-regulation of Ca<sup>2<bold>+</bold></sup> influx during repeated membrane depolarization. CDF of L-type Ca<sup>2<bold>+</bold></sup> channels (Ca<sub>v</sub>1.2) contributes to the positive force–frequency effect in the heart and is believed to involve the activation of Ca<sup>2<bold>+</bold></sup>/calmodulin-dependent kinase II (CaMKII). How CaMKII is activated and what its substrates are have not yet been determined. We show that the pore-forming subunit α<sub>1C</sub> (Ca<sub>v</sub>α1.2) is a CaMKII substrate and that CaMKII interaction with the COOH terminus of α<sub>1C</sub> is essential for CDF of L-type channels. Ca<sup>2<bold>+</bold></sup> influx triggers distinct features of CaMKII targeting and activity. After Ca<sup>2<bold>+</bold></sup>-induced targeting to α<sub>1C</sub>, CaMKII becomes tightly tethered to the channel, even after calcium returns to normal levels. In contrast, activity of the tethered CaMKII remains fully Ca<sup>2<bold>+</bold></sup>/CaM dependent, explaining its ability to operate as a calcium spike frequency detector. These findings clarify the molecular basis of CDF and demonstrate a novel enzymatic mechanism by which ion channel gating can be modulated by activity.</p></abstract></article-meta><notes><fn-group><fn><p>A. Hudmon's present address is Dept. of Neurology, Yale University, New Haven, CT 06516.</p></fn><fn><p>H. Schulman's present address is SurroMed, Inc., Menlo Park, CA 94025.</p></fn><fn><p>Abbreviations used in this paper: AIP-2, autocamtide-2–related inhibitory peptide; AKAP, A-kinase anchor protein; CaMKII, Ca<sup>2+</sup>/calmodulin-dependent kinase II; CDF, Ca<sup>2+</sup>-dependent facilitation; CDI, Ca<sup>2+</sup>-dependent inactivation; HEK, human embryonic kidney; NMDAR, <italic>N</italic>-methyl-<sc>d</sc>-aspartate receptor; PKA, protein kinase A; <italic>P<sub>o</sub></italic>, open probability; PP1, protein phosphatase 1.</p></fn></fn-group></notes></front><body><sec><title>Introduction</title><p>Ca<sup>2+</sup>-dependent facilitation (CDF) of calcium channels serves to potentiate the Ca<sup>2+</sup> influx through the L-type channels during repeated activity. CDF is a feed-forward form of adaptive plasticity that is a critical regulatory feature of many excitable cells. In the heart, frequency-dependent potentiation of Ca<sup>2+</sup> current through L-type channels (Ca<sub>v</sub>1.2; <xref rid="bib52" ref-type="bibr">Noble and Shimoni, 1981</xref>; <xref rid="bib46" ref-type="bibr">Marban and Tsien, 1982</xref>; <xref rid="bib40" ref-type="bibr">Lee, 1987</xref>; <xref rid="bib60" ref-type="bibr">Schouten and Morad, 1989</xref>; <xref rid="bib73" ref-type="bibr">Zygmunt and Maylie, 1990</xref>) contributes to the force–frequency relationship of cardiac contraction (<xref rid="bib36" ref-type="bibr">Koch-Weser and Blinks, 1963</xref>). This increased contraction strength with faster heart rates contributes to the positive inotropic response during exercise (<xref rid="bib57" ref-type="bibr">Ross et al., 1995</xref>) and is abnormal in heart failure (<xref rid="bib22" ref-type="bibr">Feldman et al., 1988</xref>; <xref rid="bib51" ref-type="bibr">Mulieri et al., 1992</xref>; <xref rid="bib28" ref-type="bibr">Hasenfuss et al., 1994</xref>). In the brain, CDF of L-type channels may be important in relation to the privileged role of L-type channels in excitation–transcription coupling (<xref rid="bib13" ref-type="bibr">Deisseroth et al., 2003</xref>). Despite these important physiological roles that are central to cardiac function and neuronal plasticity, there is little understanding of the molecular mechanism of CDF of L-type channels.</p><p>Ca<sup>2+</sup>/calmodulin-dependent protein kinase II (CaMKII), a multifunctional Ser/Thr protein kinase, is a likely effector of CDF. Pharmacological inhibition of CaMKII abolishes CDF in the heart (<xref rid="bib68" ref-type="bibr">Xiao et al., 1994</xref>; <xref rid="bib69" ref-type="bibr">Yuan and Bers, 1994</xref>). Addition of activated CaMKII to the cytoplasmic face of cardiac myocyte membranes induces a high open-probability state of the channel that is consistent with the properties of Ca<sup>2+</sup> channels displaying CDF (<xref rid="bib18" ref-type="bibr">Dzhura et al., 2000</xref>). Further, immunocytochemical data suggest that the Ca<sub>v</sub>1.2 and CaMKII are localized close to each other on the cardiomyocyte sarcolemmal membrane (<xref rid="bib68" ref-type="bibr">Xiao et al., 1994</xref>), suggesting that the kinase has easy access to the channel.</p><p>CaMKII has structural and functional properties that make it an ideal candidate to sense the frequency of Ca<sup>2+</sup> transients during neuronal firing or changes in cardiac rhythm and translate that frequency signal into activity-dependent alterations such as CDF. CaMKII is a multimeric holoenzyme composed of 12 subunits, with the subunit isoforms being derived from a family of four closely related genes (α, β, γ, and δ; <xref rid="bib32" ref-type="bibr">Hudmon and Schulman, 2002b</xref>). In the brain, α-CaMKII has been shown to play a key role in synaptic plasticity and learning/memory (<xref rid="bib43" ref-type="bibr">Lisman et al., 2002</xref>). The γ and δ isoforms predominate in the heart and have been implicated in the regulation of gene expression as well as CDF (<xref rid="bib70" ref-type="bibr">Zhang et al., 2002</xref>). In all of these isoforms, activation proceeds by Ca<sup>2+</sup>/CaM binding to an autoregulatory region, which causes the removal of a pseudosubstrate domain from the catalytic site. After the initial stimulus, autophosphorylation of Thr<sup>286</sup> or its equivalent (Thr<sup>287</sup> in non–α isoforms) renders subsequent kinase activity independent (autonomous) of Ca<sup>2+</sup> and CaM (<xref rid="bib50" ref-type="bibr">Miller et al., 1988</xref>) and increases the kinase's affinity for CaM by >10,000-fold (“CaM trapping”; <xref rid="bib49" ref-type="bibr">Meyer et al., 1992</xref>). These properties endow CaMKII with the ability to become persistently activated in a transition that is sharply dependent on the frequency of Ca<sup>2+</sup> oscillations (<xref rid="bib12" ref-type="bibr">De Koninck and Schulman, 1998</xref>; <xref rid="bib21" ref-type="bibr">Eshete and Fields, 2001</xref>; <xref rid="bib3" ref-type="bibr">Bayer et al., 2002</xref>; <xref rid="bib7" ref-type="bibr">Bradshaw et al., 2003</xref>).</p><p>We now demonstrate for the first time that the subcellular localization of CaMKII is critical for its biological role as a frequency decoder of voltage-driven calcium spikes. We show that CaMKII phosphorylates α<sub>1C</sub> and that tethering of CaMKII to the α<sub>1C</sub> COOH terminus is an essential molecular feature of CDF. We present a molecular model for CDF in which a dedicated CaMKII holoenzyme acts as both a local sensor to monitor Ca<sup>2+</sup> channel activity and as a resident kinase effector to regulate Ca<sup>2+</sup> channel activity.</p></sec><sec><title>Results</title><sec><title>The NH<sub>2</sub> and COOH termini of α<sub>1C</sub> are substrates of CaMKII</title><p>Modulation of L-type channel gating by cytoplasmic delivery of constitutively active CaMKII is blocked by nonhydrolyzable analogues of ATP (<xref rid="bib18" ref-type="bibr">Dzhura et al., 2000</xref>), suggesting that the kinase acts through phosphorylation of the channel or an associated regulatory protein. Because the kinase-induced increase in L-type Ca<sup>2+</sup> current by both protein kinase A (PKA) and Src results from phosphorylation of α<sub>1C</sub> (<xref rid="bib11" ref-type="bibr">De Jongh et al., 1996</xref>; <xref rid="bib4" ref-type="bibr">Bence-Hanulec et al., 2000</xref>), we first tested whether α<sub>1C</sub> was also a substrate for activated CaMKII (<xref rid="fig1" ref-type="fig">Fig. 1</xref> A). The addition of activated CaMKII to α<sub>1C</sub> immunoprecipitated from lysates of L-type channel–expressing human embryonic kidney (HEK) cells resulted in the phosphorylation of protein migrating at ∼240 kD, consistent with the molecular mass of α<sub>1C</sub>. The kinase activity could be attributed to CaMKII and not to another kinase coimmunoprecipitated with α<sub>1C</sub> because inclusion of the CaMKII inhibitor autocamtide-2–related inhibitory peptide (AIP-2) prevented phosphorylation; continued presence of the α<sub>1C</sub> protein under this condition was confirmed by immunoblotting (<xref rid="fig1" ref-type="fig">Fig. 1</xref> A, bottom). The immunoprecipitated and phosphorylated protein could be confidently identified as α<sub>1C</sub> in light of the findings that no α<sub>1C</sub> was immunoprecipitated and that <sup>32</sup>P was not incorporated when immunoprecipitation was performed with control IgG or with lysates of HEK cells in which α<sub>1C</sub> had not been expressed. Interestingly, under conditions in which α<sub>1C</sub> was phosphorylated by CaMKII (<xref rid="fig1" ref-type="fig">Fig. 1</xref> A, lane 3), we noticed a <sup>32</sup>P-labeled protein (∼50 kD) corresponding to the autophosphorylated form of the α subunit of CaMKII that had been introduced for the kinase assay. Immunoblots with an anti-CaMKII antibody confirmed its identity (not depicted, but see <xref rid="fig5" ref-type="fig">Fig. 5</xref> E). The retention of CaMKII, despite extensive washing of the immobilized α<sub>1C</sub>, suggested that α<sub>1C</sub> may serve as an anchoring protein as well as a substrate for the kinase. The near absence of retention when AIP-2 was added to the reaction gave an early indication about the mechanism of anchoring (see <xref rid="fig6" ref-type="fig">Fig. 6</xref> B).</p><fig id="fig1" position="float"><label>Figure 1.</label><caption><p>
<bold>Phosphorylation of the α<sub>1C</sub> subunit by CaMKII.</bold> (A) Autoradiogram showing phosphorylation of α<sub>1C</sub> by CaMKII. Lysates from HEK cells transfected with α<sub>1C</sub> and β2b (lanes 2–4) or nontransfected cells (lane 1) were immunoprecipitated with an anti-α<sub>1C</sub> antibody (lanes 1, 3, and 4) or control IgG (lane 2) and then incubated with purified α-CaMKII in the presence of Ca<sup>2+</sup>/CaM and Mg<sup>2+</sup>/ATP<sup>32</sup> as described in Materials and methods. 200 nM of the CaMKII inhibitor peptide AIP-2 (Calbiochem) was included (lane 4) to demonstrate kinase specificity. Phosphorylated α<sub>1C</sub> is indicated by an arrowhead; autophosphorylated CaMKII, retained after the kinase reaction despite extensive washing of the immunoprecipitate, is indicated with a double arrowhead. An anti-α<sub>1C</sub> immunoblot of the samples used in the kinase reaction is shown below the autoradiogram. (B) Schematic of α<sub>1C</sub>. Thick black lines highlight regions used to generate GST fusion proteins. (C) GST fusion proteins enriched from bacterial lysates using glutathione–sepharose were incubated with purified α-CaMKII in the presence of Ca<sup>2+</sup>/CaM and Mg<sup>2+</sup>/ATP<sup>32</sup> as described in Materials and methods. After extensive washes, proteins were eluted using SDS-PAGE sample buffer. Autoradiogram of fusion proteins separated by SDS-PAGE after phosphorylation by CaMKII. C-term refers to the more distal COOH-terminal fusion protein, containing aa 1669–2171. Above the autoradiogram is the Coomassie blue–stained band for each fusion protein, indicating nearly equal loading of substrate for all fusion proteins.</p></caption><graphic xlink:href="200505155f1"/></fig><p>Having demonstrated that α<sub>1C</sub> was a CaMKII substrate, we next ran tests to determine which of the intracellular domains of α<sub>1C</sub> were phosphorylated by CaMKII. GST fusion proteins were generated for the entire sequence of each of the intracellular domains of the α<sub>1C</sub> subunit except the large cytoplasmic tail, which was represented by two complementary fragments (aa 1507–1622 and 1669–2171; <xref rid="fig1" ref-type="fig">Fig. 1</xref> B). When the fusion proteins were tested in an in vitro kinase assay, significant incorporation of <sup>32</sup>P was only observed for the NH<sub>2</sub>-terminal construct and the COOH-terminal fusion protein containing aa 1669–2171 (<xref rid="fig1" ref-type="fig">Fig. 1</xref> C) and not the fusion protein containing aa 1507–1622 (not depicted). The finding that CaMKII can phosphorylate NH<sub>2</sub>- and COOH-terminal regions of α<sub>1C</sub> is provocative in light of previous data suggesting that these regions may be targets of kinase action for modulation of Ca<sub>v</sub>1.2 function (<xref rid="bib58" ref-type="bibr">Rotman et al., 1995</xref>; <xref rid="bib4" ref-type="bibr">Bence-Hanulec et al., 2000</xref>; <xref rid="bib48" ref-type="bibr">McHugh et al., 2000</xref>). Similar to results with the intact channel (<xref rid="fig1" ref-type="fig">Fig. 1</xref> A), we again noticed in multiple lanes an ∼50-kD <sup>32</sup>P-labeled protein corresponding to the autophosphorylated form of the α subunit of CaMKII that had been introduced for the kinase assay. The finding that α-CaMKII could be retained by individual domains of α<sub>1C</sub> suggested that these domains might contribute to the kinase anchoring to the channel subunit as a whole.</p></sec><sec><title>CaMKII interacts specifically with α<sub>1C</sub>
</title><p>We tested the possibility that CaMKII tethers to α<sub>1C</sub> in the rat heart by attempting to coimmunoprecipitate CaMKII with α<sub>1C</sub> (<xref rid="fig2" ref-type="fig">Fig. 2</xref> A). An anti-α<sub>1C</sub> antibody (but not control IgG) coimmunoprecipitated an ∼58-kD protein from a rat heart that was easily detectable with a biotinylated calmodulin overlay, which was consistent with the properties of δ-CaMKII. Tethering of the kinase to the pore-forming subunit was further evaluated in experiments with HEK 293 cells coexpressing GFP-tagged CaMKII and Xpress-tagged α<sub>1C</sub>, along with the calcium channel accessory subunits α<sub>2</sub>δ and β<sub>2</sub> (<xref rid="fig2" ref-type="fig">Fig. 2</xref> B). We observed coimmunoprecipitation of the GFP-CaMKII by the antibody to epitope-tagged α<sub>1C</sub> (<xref rid="fig2" ref-type="fig">Fig. 2</xref> B, lane 5), but not by a control IgG (lane 4).</p><fig id="fig2" position="float"><label>Figure 2.</label><caption><p>
<bold>CaMKII coimmunoprecipitates and colocalizes with α<sub>1C</sub>.</bold> (A) Biotinylated calmodulin overlay of rat cardiac sarcolemmal membranes after immunoprecipitation with an anti-α<sub>1C</sub> antibody. Purified α-CaMKII was run as a control to demonstrate effectiveness of CaM overlay. An anti-α<sub>1C</sub> antibody (but not control IgG) coimmunoprecipitated a protein identified as the δ isoform of CaMKII by biotinylated CaM overlay and apparent molecular mass. (B) Anti-GFP immunoblot after immunoprecipitation of GFP-CaMKII by control IgG (lane 4) or anti-α<sub>1C</sub> antibody (lane 5) from lysates of HEK 293 cells transiently transfected with GFP-CaMKII and α<sub>1C</sub>.</p></caption><graphic xlink:href="200505155f2"/></fig></sec><sec><title>Activity-dependent association of CaMKII with multiple cytoplasmic regions of α<sub>1C</sub>
</title><p>To further define the interaction between CaMKII and α<sub>1C</sub>, we constructed a pull-down binding assay using various α<sub>1C</sub>-GST fusion proteins (<xref rid="fig3" ref-type="fig">Fig. 3</xref>). The goal was to find out whether a direct interaction could be observed in vitro and whether different activation states of CaMKII modulated binding. When CaMKII was activated with Ca<sup>2+</sup>/CaM but not allowed to undergo autophosphorylation (ATP not included), the kinase bound to the NH<sub>2</sub>-terminal domain and the III-IV loop of α<sub>1C</sub> (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, middle). Subsequent removal of Ca<sup>2+</sup>/CaM in the wash buffer reversed this binding (unpublished data). When CaMKII was activated in the presence of Ca<sup>2+</sup>/CaM plus ATP on ice, conditions previously shown to produce predominantly Thr<sup>286</sup> autophosphorylation (<xref rid="bib38" ref-type="bibr">Lai et al., 1987</xref>; <xref rid="bib44" ref-type="bibr">Lou and Schulman, 1989</xref>; <xref rid="bib34" ref-type="bibr">Ikeda et al., 1991</xref>), CaMKII again bound to the NH<sub>2</sub> terminus and III-IV loop, but additionally bound to the I-II loop and the COOH terminus (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, bottom). In contrast, CaMKII did not bind to any of the cytoplasmic region–containing GST fusion proteins in the absence of activating stimuli (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, top). We concluded that the initiation of a direct interaction between CaMKII and α<sub>1C</sub> requires activation of the kinase by Ca<sup>2+</sup>/CaM. A subsequent activation state, that produced by autophosphorylation, was necessary for binding to additional cytoplasmic regions of α<sub>1C</sub>.</p><fig id="fig3" position="float"><label>Figure 3.</label><caption><p>
<bold>Activity-dependent interaction of CaMKII with the cytoplasmic determinants of α<sub>1C</sub>.</bold> Immunoblots using an mAb (CBα2) for CaMKII after a GST pull-down assay with 20 nM of native (top), Ca<sup>2+</sup>/CaM-activated (middle), or Ca<sup>2+</sup>/CaM/autophosphorylated α-CaMKII (bottom). GST fusion proteins contained various cytoplasmic regions of α<sub>1C</sub> just as in <xref rid="fig1" ref-type="fig">Fig. 1</xref> C. Panel above the immunoblots shows a representative Ponceau stain of each fusion protein. Although only one Ponceau staining profile is shown, all blots were run in parallel, and equal loading of all fusions proteins was independently verified.</p></caption><graphic xlink:href="200505155f3"/></fig><p>To identify novel structural determinants of α<sub>1C</sub> that functionally affect CDF, we initially focused on the COOH terminus. This region displays an appropriate combination of attributes for CaMKII-mediated CDF: it is a target for phosphorylation by CaMKII (<xref rid="fig1" ref-type="fig">Fig. 1</xref> C); it binds preferentially to autophosphorylated CaMKII (<xref rid="fig3" ref-type="fig">Fig. 3</xref>), a state of the kinase capable of supporting facilitation of single channels (<xref rid="bib18" ref-type="bibr">Dzhura et al., 2000</xref>); and it has been implicated in Ca<sup>2+</sup>-dependent modulation of channel function (<xref rid="bib29" ref-type="bibr">Hell et al., 1995</xref>; <xref rid="bib71" ref-type="bibr">Zühlke et al., 1999</xref>; <xref rid="bib24" ref-type="bibr">Gao et al., 2001</xref>). To delimit the locus of CaMKII binding within the COOH-terminal tail of α<sub>1C</sub>, we used a series of GST fusion proteins corresponding to different portions of this region (<xref rid="fig4" ref-type="fig">Fig. 4</xref> A). We found a pattern of interactions with autophosphorylated CaMKII that suggested that the kinase bound between residues 1622 and 1669 of α<sub>1C</sub>. Because a weak interaction was also seen with a construct proximal to 1622, we generated a fusion protein spanning aa 1581–1690 for additional testing and found a clear interaction (<xref rid="fig4" ref-type="fig">Fig. 4</xref> A). To further narrow down the CaMKII interaction site within this 110-aa region, we probed its interaction with autophosphorylated CaMKII and assessed interference by a series of ∼22 overlapping aa peptides (<xref rid="fig4" ref-type="fig">Fig. 4</xref> B; <xref rid="bib55" ref-type="bibr">Pitt et al., 2001</xref>). A peptide generated from residues 1639–1660 dramatically reduced the interaction of the kinase with the 1581–1690 fusion protein. In contrast, the CaMKII interaction was not inhibited by two peptides (1589–1610 and 1615–1636) that corresponded to sites important for tethering of apoCaM (<xref rid="bib55" ref-type="bibr">Pitt et al., 2001</xref>; <xref rid="bib35" ref-type="bibr">Kim et al., 2004</xref>). One stretch of six residues within the 1639–1660 peptide, TVGKF(<sup>Y</sup>/<sub>I</sub>)A, was identified as being nearly identical in α<sub>1C</sub> and α<sub>1A</sub> (Ca<sub>v</sub>2.1), the pore-forming subunit of P/Q-type Ca<sup>2+</sup> channels, which display their own form of CDF (<xref rid="bib39" ref-type="bibr">Lee et al., 1999</xref>; <xref rid="bib14" ref-type="bibr">DeMaria et al., 2001</xref>). Accordingly, we constructed an α<sub>1C</sub> fusion protein containing the amino acids EEDAAA in place of TVGKFY within an otherwise wild-type sequence of residues 1581–1690 (Mut6). CaMKII binding to the Mut6 fusion protein was reduced by 87.3 ± 4.5% relative to binding to wild-type 1581–1690 fusion protein (<xref rid="fig4" ref-type="fig">Fig. 4</xref> C). In contrast, the same amino acid substitution left CaM binding to this mutant fusion protein unaffected (<xref rid="fig4" ref-type="fig">Fig. 4</xref> D).</p><fig id="fig4" position="float"><label>Figure 4.</label><caption><p>
<bold>Localization of the CaMKII binding site on the COOH terminus of α<sub>1C</sub>.</bold> (A) Diagram of α<sub>1C</sub> fusion proteins used in GST pull-down assays with autophosphorylated α-CaMKII, exhibiting robust (+), partial (±), and no (−) binding. (B) Immunoblot with CBα2 after GST pull down of 20 nM of purified autophosphorylated α-CaMKII, using α<sub>1C</sub> aa 1581–1690 fused to GST. Pull-down assay performed in the presence of 40 μM of the indicated peptide or the peptide diluent DMSO. (C) Quantification after immunoblot with CBα2 of GST pull-down assays of purified autophosphorylated α-CaMKII, using α<sub>1C</sub> aa 1581–1690 (wild type [WT]), a <sup>1644</sup>TVGKFY<sup>1649</sup> → EEDAAA mutant (Mut6), or GST alone shows that Mut6 blocks CaMKII binding. Panel above the immunoblots shows a representative Ponceau stain of each fusion protein. *, P < 0.001 for a one-way analysis of variance followed by Dunnett's test to identify specific pair-wise differences between the means for Mut6 versus WT and GST versus WT (<italic>n</italic> = 4–8). Inset shows an exemplar immunoblot with CBα2. (D) An exemplar immunoblot with an anti-CaM antibody, showing that CaM binding is not affected by the Mut6 mutation. Panel above the immunoblots shows a representative Ponceau stain of each fusion protein.</p></caption><graphic xlink:href="200505155f4"/></fig></sec><sec><title>Disruption of CaMKII binding to the COOH terminus of α<sub>1C</sub> prevents CDF</title><p>We then tested whether this site was critical for CDF by introducing the Mut6 mutation into α<sub>1C</sub> subunits of L-type channels expressed in <italic>Xenopus laevis</italic> oocytes. Because L-type channels also display a strong Ca<sup>2+</sup>-dependent inactivation (CDI) process that could diminish our ability to detect facilitation, we sought conditions under which CDF could be observed without the counteraction of CDI. Fortunately, robust CDF during trains of depolarizing pulses can be obtained by means of a point mutation within the IQ motif (I1654A; <xref rid="bib71" ref-type="bibr">Zühlke et al., 1999</xref>, <xref rid="bib72" ref-type="bibr">2000</xref>) that eliminates CDI. In this setting, the Mut6 modification of the CaMKII interaction site completely abolished CDF (<xref rid="fig5" ref-type="fig">Fig. 5</xref>, A–C). There was no potentiation of <italic>I</italic>
<sub>Ca</sub> at any point during the train of 40 successive depolarizations within the entire range of frequencies tested (0.5–3.3 Hz). Abolition of the Ca<sup>2+</sup>-dependent facilitatory process was also observed in experiments using a two-pulse protocol and finely graded changes in interpulse interval. Ca<sup>2+</sup> currents evoked by the second pulse averaged 110% of those elicited by the first pulse at a time interval when the peak Ba<sup>2+</sup> current had only recovered to ∼95% (<xref rid="fig5" ref-type="fig">Fig. 5</xref> D). A comparable difference between recovery of Ca<sup>2+</sup> and Ba<sup>2+</sup> currents was seen in wild-type α<sub>1C</sub> (<xref rid="bib71" ref-type="bibr">Zühlke et al., 1999</xref>, <xref rid="bib72" ref-type="bibr">2000</xref>) but was likewise abolished by the Mut6 modification (unpublished data). Thus, in both of the approaches used to assess facilitation—potentiation of <italic>I</italic>
<sub>Ca</sub> during trains of depolarizations and recovery from the aftereffects of a single pulse—CDF was abolished by mutation of the COOH-terminal CaMKII interaction site on Ca<sub>v</sub>1.2.</p><fig id="fig5" position="float"><label>Figure 5.</label><caption><p>
<bold>CaMKII interaction with the COOH terminus of α<sub>1C</sub> is essential for CDF.</bold> (A) <italic>I</italic>
<sub>Ba</sub> and scaled <italic>I</italic>
<sub>Ca</sub> traces during a train of 40 test pulses of <italic>V</italic>
<sub>h</sub> from –90 mV to +20 mV at 3.3 Hz recorded from oocytes expressing α<sub>1C</sub> I1654A (I/A) or α<sub>1C</sub> I1654A/<sup>1644</sup>TVGKFY<sup>1649</sup> → EEDAAA (I/A-Mut6). Bars, 500 nA and 25 ms. (B) Peak <italic>I</italic>
<sub>Ba</sub> and <italic>I</italic>
<sub>Ca</sub> during trains of 40 repetitive test pulses at 3.3 Hz, normalized to the current amplitude at the beginning of each train (<italic>n</italic> = 4–5). Values indicate means ± SEM. (C) Changes in peak <italic>I</italic>
<sub>Ba</sub> and <italic>I</italic>
<sub>Ca</sub> conducted by α<sub>1C</sub> I1654A (I/A) or α<sub>1C</sub> I1654A/<sup>1644</sup>TVGKFY<sup>1649</sup> → EEDAAA (I/A-Mut6) at indicated stimulation frequencies (<italic>n</italic> = 4–5) Values indicate means ± SEM. (D) Summary of the recovery from inactivation after a two-step protocol for I/A and I/A-Mut6. The length of the prepulse was individually determined for each oocyte to produce ∼75–90% inactivation. (E) Autoradiograph showing phosphorylation of wild type (WT) or mutant α<sub>1C</sub> (Mut6) by CaMKII, performed as in <xref rid="fig1" ref-type="fig">Fig. 1</xref> A. An anti-α<sub>1C</sub> immunoblot of the samples used in the kinase reaction confirmed similar expression levels of the WT and mutant α<sub>1C</sub> subunits. An anti-CaMKII immunoblot with CBα2 confirmed the identity of the retained 50-kD <sup>32</sup>P-labeled protein as α-CaMKII.</p></caption><graphic xlink:href="200505155f5"/></fig><p>To examine the mechanism by which the mutation prevented CDF, we tested whether the Mut6 channel was still a substrate for CaMKII using an in vitro assay like that in <xref rid="fig1" ref-type="fig">Fig. 1</xref>, in which the availability of kinase for phosphorylation was not limited and not dependent on tethering to the COOH terminus. Disruption of the CaMKII binding site on the COOH terminus by the Mut6 substitution did not reduce <sup>32</sup>P incorporation into α<sub>1C</sub> (<xref rid="fig5" ref-type="fig">Fig. 5</xref> E), suggesting that the α<sub>1C</sub> retained its ability to undergo phosphorylation. Together, these data support the hypothesis that CDF depends on tethering of CaMKII to this COOH-terminal site. Like the wild-type α<sub>1C</sub>, the Mut6 α<sub>1C</sub> displayed an interaction with autophosphorylated (<sup>32</sup>P-labeled) CaMKII. The retention of kinase binding was not surprising in light of the multiple sites on α<sub>1C</sub> for CaMKII interaction that we had previously identified (<xref rid="fig3" ref-type="fig">Fig. 3</xref>).</p></sec><sec><title>The CaMKII binding site for the COOH terminus of α<sub>1C</sub> is conserved among multiple CaMKII isoforms and localizes to the catalytic domain</title><p>Although we had used α-CaMKII, the predominantly brain-enriched isoform studied in the preceding in vitro experiments, there are several other CaMKII isoforms that differ in their cellular and subcellular distributions (<xref rid="bib31" ref-type="bibr">Hudmon and Schulman, 2002a</xref>). The δ isoform, the major CaMKII isoform in the heart (<xref rid="bib19" ref-type="bibr">Edman and Schulman, 1994</xref>), was of particular interest (<xref rid="fig2" ref-type="fig">Fig. 2</xref> A). Accordingly, we examined the generality of CaMKII interactions with the COOH-terminal tail of α<sub>1C</sub> across a range of isoforms. The α, β, γ<sub>B</sub>, δ<sub>A</sub>, and δ<sub>C</sub> isoforms were transiently expressed in HEK 293 cells for use as source material in pull-down assays and detected by the sensitive calmodulin overlay technique (<xref rid="bib25" ref-type="bibr">Glenney and Weber, 1983</xref>; <xref rid="fig6" ref-type="fig">Fig. 6</xref> A). In the absence of autophosphorylation, no binding was ever observed for any of the isoforms tested (unpublished data). However, once autothiophosphorylated, robust binding to the α<sub>1C</sub> COOH-terminal tail was observed for each of these CaMKII isoforms, with the sole exception of γ<sub>B</sub>-CaMKII. Thus, the capability of interaction with Ca<sub>v</sub>1.2 is a widespread property of the CaMKII family, including the α/β and δ isoforms prevalent in brain and cardiac tissue.</p><fig id="fig6" position="float"><label>Figure 6.</label><caption><p>
<bold>The binding site for the COOH terminus of α<sub>1C</sub> on CaMKII is localized near the catalytic domain.</bold> (A) Biotinylated CaM overlay of GST pull downs, using a fusion protein from the COOH terminus of α<sub>1C</sub> (aa 1509–1905) on lysates of HEK 293 cells transiently transfected with the CaMKII isoforms (α, β, δ<sub>A</sub>, δC, and γ<sub>B</sub>; arrows) after thioautophosphorylation. In lanes 6 and 7, lysates of untransfected cells were run with (+) and without (−) purified thiophosphorylated α-CaMKII added to the lysate. (B) Immunoblot using an mAb (CBα2) for CaMKII after GST pull downs, using a fusion protein from the COOH terminus of α<sub>1c</sub> (aa 1509–1905) and 20 nM of purified autophosphorylated α-CaMKII. In addition, 20 μM of the indicated peptide was added to each binding reaction. (C) Sequence alignment of CaMKII binding sites from the COOH termini of NR2B and α<sub>1C</sub> with the autoregulatory domain from α-CaMKII.</p></caption><graphic xlink:href="200505155f6"/></fig><p>Where is the binding site for α<sub>1C</sub> on CaMKII? The conserved nature of the α<sub>1C</sub> binding site between brain and cardiac CaMKII isoforms favored a binding site that is conserved among the different kinase isoforms. We examined the conserved catalytic domain of α-CaMKII, based on a recent report describing its interaction with the COOH terminus of the NR2B subunit of the neuronal <italic>N</italic>-methyl-<sc>d</sc>-aspartate receptor (NMDAR; <xref rid="bib2" ref-type="bibr">Bayer et al., 2001</xref>). Indeed, binding of the COOH-terminal tail of α<sub>1C</sub> to autophosphorylated CaMKII was blocked by a peptide modeled after the CaMKII binding site of the NR2B subunit (NR2B peptide; <xref rid="fig6" ref-type="fig">Fig. 6</xref> B). Further, binding of α<sub>1C</sub> to CaMKII was potently blocked by peptides designed around Thr<sup>286</sup> and the autoregulatory domain of CaMKII, including the peptide substrate AC-2 and the peptide inhibitor AC-3i (<xref rid="fig6" ref-type="fig">Fig. 6</xref> B), as well as AIP-2 (<xref rid="fig1" ref-type="fig">Fig. 1</xref> A). As expected, the control peptide AC-3c had no effect on binding. Both sets of observations resemble previous findings using peptide inhibition to study binding of CaMKII to NR2B (<xref rid="bib63" ref-type="bibr">Strack et al., 2000</xref>; <xref rid="bib2" ref-type="bibr">Bayer et al., 2001</xref>). A logical conclusion is that similar or identical molecular determinants on CaMKII are responsible for binding either to α<sub>1C</sub> or to NR2B. The NR2B sequence that was found to support interaction with CaMKII closely resembles the autoregulatory domain of CaMKII surrounding Thr<sup>286</sup> (<xref rid="fig6" ref-type="fig">Fig. 6</xref> C; <xref rid="bib2" ref-type="bibr">Bayer et al., 2001</xref>). In turn, both of these stretches of amino acids show significant resemblance to the region of α<sub>1C</sub> that we identified as critical for CaMKII interaction by peptide competition (<xref rid="fig4" ref-type="fig">Fig. 4</xref> B), and that includes the TVGKFY sequence that was altered to the detriment of the α<sub>1C</sub>–CaMKII interaction. Although the corresponding regions of α<sub>1C</sub> and NR2B display points of sequence similarity (<xref rid="fig6" ref-type="fig">Fig. 6</xref> C, dots and dashes), the overall degree of homology is limited.</p></sec><sec><title>CaMKII binding to the COOH terminus of α<sub>1C</sub> produces a dedicated Ca<sup>2+</sup> sensor</title><p>The functional nature of the channel–kinase interaction could follow one of several possible scenarios. During recurrent rises and falls in Ca<sup>2+</sup>, the enzyme might cycle on and off the channel. Alternatively, CaMKII might remain anchored to α<sub>1C</sub> with its activity persistently switched on, like CaMKII associated with the NMDAR (<xref rid="bib2" ref-type="bibr">Bayer et al., 2001</xref>). Finally, CaMKII might stay tethered to the α<sub>1C</sub> subunit, like PKA associated with Ca<sub>v</sub>1.2 through A-kinase anchor protein (AKAP; <xref rid="bib64" ref-type="bibr">Tavalin et al., 1999</xref>), but with kinase activity modulated by local changes in Ca<sup>2+</sup>/CaM, similar to the way that PKA is regulated by cAMP for β-adrenergic modulation (<xref rid="bib23" ref-type="bibr">Gao et al., 1997</xref>). To explore these possibilities, we tested whether CaMKII dissociated from the COOH-terminal tail on reversal of the Ca<sup>2+</sup> elevation or the kinase activation that initially drove the interaction.</p><p>When the Ca<sup>2+</sup> chelator EGTA was added immediately after the preautophosphorylation reaction, the binding of CaMKII to the α<sub>1C</sub> COOH-terminal tail was inhibited (<xref rid="fig7" ref-type="fig">Fig. 7</xref> A). In contrast, once autophosphorylated CaMKII had bound to the α<sub>1C</sub> COOH-terminal tail, EGTA in the wash buffer (two or three rounds of washing, each lasting ∼5 min) failed to dissociate the kinase (<xref rid="fig7" ref-type="fig">Fig. 7</xref> A). Dephosphorylation of autophosphorylated CaMKII with protein phosphatase 1 (PP1) before presenting the kinase to the α<sub>1C</sub> COOH-terminal fusion protein prevented binding (<xref rid="fig7" ref-type="fig">Fig. 7</xref> B). However, dephosphorylation of CaMKII after binding did not. Even the combination of postbinding dephosphorylation and EGTA application failed to reverse binding (<xref rid="fig7" ref-type="fig">Fig. 7</xref> B). In control experiments, immunoblotting with the phosphospecific antibody indicated that Thr<sup>286</sup> had been completely dephosphorylated by PP1 treatment after the initial kinase binding (<xref rid="fig7" ref-type="fig">Fig. 7</xref> B). Thus, although Ca<sup>2+</sup>/CaM and autophosphorylation were necessary for CaMKII to bind to the α<sub>1C</sub> COOH terminus, the same conditions were no longer required to sustain the interaction.</p><fig id="fig7" position="float"><label>Figure 7.</label><caption><p>
<bold>CaMKII interaction with the COOH terminus of α<sub>1C</sub> is not reversed by dephosphorylation or CaM dissociation, and tethered CaMKII requires autophosphorylation or Ca<sup>2</sup></bold>
<sup><bold>+</bold></sup>
<bold>/CaM for activity.</bold> (A and B) Immunoblots with CBα2 or a phosphospecific CaMKII mAb after GST pull-down assays, using α<sub>1C</sub> aa 1509–1905 and 20 nM of autophosphorylated α-CaMKII. (A) 5 mM EGTA was present in the binding reaction and/or in the wash. (B) Purified recombinant PP1 was added before (PP1-Pre) or after (PP1-Post) the binding reaction in the presence or absence of 5 mM EGTA, as indicated. (C) Time course of reversal of CaMKII autonomous activity after PP1 treatment in solution (<italic>n</italic> = 4). (D) Activity measurements, using peptide AC-2 as a substrate, of CaMKII recovered in GST pull-down assays, using α<sub>1C</sub> aa 1509–1905. Ca<sup>2+</sup>/CaM-dependent and autonomous activity measurements of CaMKII recovered after treatment with recombinant PP1 for 30 min (PP1) or no treatment (−) in the binding assay (<italic>n</italic> = 4) Values indicate means ± SD.</p></caption><graphic xlink:href="200505155f7"/></fig></sec><sec><title>Tethered CaMKII retains its dependence on Ca<sup>2+</sup>/CaM for activity</title><p>Because the CaMKII binding for both α<sub>1C</sub> and NR2B appears to localize to the catalytic domain of the kinase, we asked whether α<sub>1C</sub> binding to CaMKII regulates its kinase activity, as in the case of NR2B. When bound to NR2B, CaMKII remains active in phosphorylating substrates even in the absence of Ca<sup>2+</sup>/CaM and autophosphorylation (<xref rid="bib2" ref-type="bibr">Bayer et al., 2001</xref>). To determine how CaMKII is regulated when it is stably bound to the α<sub>1C</sub> COOH terminus, we examined the Ca<sup>2+</sup>/CaM-dependent and -independent (autonomous) activity after PP1 treatment. Dephosphorylation by PP1, assessed by tracking the loss of autonomous activity for soluble kinase, was complete within 30 min (<xref rid="fig7" ref-type="fig">Fig. 7</xref> C). Under similar conditions, we observed that treatment of α<sub>1C</sub>-bound kinase with PP1 completely eliminated autonomous activity (remaining activity was 1.2 ± 0.6% of that without PP1 treatment; <xref rid="fig7" ref-type="fig">Fig. 7</xref> D). Thus, autonomous activity of bound CaMKII was not maintained merely by interaction of the kinase with the α<sub>1C</sub> COOH terminus but depended strictly on CaMKII autophosphorylation. After PP1 treatment, tethered CaMKII could be reactivated by Ca<sup>2+</sup>/CaM. In these respects, CaMKII binding to α<sub>1C</sub> or to NR2B had very different effects on the activity of the kinase. As mentioned in Discussion, the association of CaMKII to the α<sub>1C</sub> COOH terminus is well suited to localize the kinase in close proximity to its regulatory target but not to keep the kinase constitutively active.</p></sec></sec><sec><title>Discussion</title><p>CDF is a powerful positive feedback mechanism that allows excitable cells such as myocytes and neurons to modulate Ca<sup>2+</sup> entry through Ca<sup>2+</sup> channels according to the previous pattern of repetitive activity. The functional consequences are clearest in the heart, where CDF of L-type channels is required for sinoatrial pacemaker activity (<xref rid="bib65" ref-type="bibr">Vinogradova et al., 2000</xref>) and contributes to the myocardial force–frequency relationship (<xref rid="bib36" ref-type="bibr">Koch-Weser and Blinks, 1963</xref>). However, CDF or related phenomena have also been described for voltage-gated Ca<sup>2+</sup> channels in neurons (<xref rid="bib10" ref-type="bibr">Cuttle et al., 1998</xref>), smooth muscle cells (<xref rid="bib47" ref-type="bibr">McCarron et al., 1992</xref>), and adrenal glomerulosa cells (<xref rid="bib67" ref-type="bibr">Wolfe et al., 2002</xref>). Although not described in neurons, CDF of L-type channels could play a major role in supporting their privileged status in mediating excitation–transcription coupling and long-term synaptic plasticity (<xref rid="bib5" ref-type="bibr">Bradley and Finkbeiner, 2002</xref>; <xref rid="bib66" ref-type="bibr">West et al., 2002</xref>; <xref rid="bib13" ref-type="bibr">Deisseroth et al., 2003</xref>).</p><p>We have presented several new findings that advance our understanding of CDF of L-type channels. First, CaMKII associates with the pore-forming α<sub>1C</sub> subunit of L-type channels in the heart as indicated by coimmunoprecipitation. Second, specific regions of the α<sub>1C</sub> subunit have the capability to directly anchor activated CaMKII. Third, CaMKII can phosphorylate α<sub>1C</sub> in regions previously implicated in regulating channel function. Fourth, a mutation in the COOH terminus of α<sub>1C</sub> that disrupted CaMKII binding to that region completely abolished CDF. Fifth, once tethered to the COOH terminus, CaMKII can be completely dephosphorylated and deactivated, even though it persists in its association and retains its dependence on Ca<sup>2+</sup>/CaM. Thus, we conclude that the localization and targeting of CaMKII to the COOH terminus of the L-type channel is critical for CDF. Our experiments suggest that individual L-type channels can take advantage of CaMKII as a frequency detector for the activity-dependent regulation of their Ca<sup>2+</sup> influx. The tethered kinase provides a local and specific integrator of preceding channel activity that controls future channel function through feed-forward autoregulation.</p><sec><title>A working model for unifying disparate observations on CDF</title><p>Our findings provide a biochemical and molecular explanation of earlier findings that suggested that CDF was mediated by CaMKII. Ca<sup>2+</sup> buffer experiments revealed that CDF depended on a calcium signal near the channel (<xref rid="bib30" ref-type="bibr">Hryshko and Bers, 1990</xref>). Pharmacological inhibition of CaMKII abolished CDF (<xref rid="bib1" ref-type="bibr">Anderson et al., 1994</xref>; <xref rid="bib68" ref-type="bibr">Xiao et al., 1994</xref>; <xref rid="bib69" ref-type="bibr">Yuan and Bers, 1994</xref>). Immunostaining showed that autophosphorylated CaMKII was concentrated near the surface membrane of cardiomyocytes (<xref rid="bib68" ref-type="bibr">Xiao et al., 1994</xref>; <xref rid="bib65" ref-type="bibr">Vinogradova et al., 2000</xref>). More recently, <xref rid="bib18" ref-type="bibr">Dzhura et al. (2000)</xref> found that direct application of thiophosphorylated (constitutively activated) CaMKII to the cytoplasmic face of cardiac myocyte membranes induced a high open probability (<italic>P<sub>o</sub></italic>) mode of L-type channel activity, thereby accounting for CDF; the modulatory effect could be prevented by nonhydrolyzable ATP analogues or CaM kinase blockers, further implicating the importance of phosphorylation by CaMKII.</p><p>Our results not only uncover key molecular underpinnings of those earlier studies but also resolve several unanswered questions. How can a ubiquitous CaMKII fulfill the requirement for a local Ca<sup>2+</sup> signal in CDF (<xref rid="bib30" ref-type="bibr">Hryshko and Bers, 1990</xref>; <xref rid="bib65" ref-type="bibr">Vinogradova et al., 2000</xref>)? Is autophosphorylated CaMKII concentrated near the cell surface (<xref rid="bib68" ref-type="bibr">Xiao et al., 1994</xref>; <xref rid="bib65" ref-type="bibr">Vinogradova et al., 2000</xref>) simply because Ca<sup>2+</sup> is highest near sites of influx (<xref rid="bib30" ref-type="bibr">Hryshko and Bers, 1990</xref>)? Is a membrane localization of CaMKII achieved by tethering to L-type channels, and is such targeting necessary for CDF? Does CaMKII mediate CDF by directly phosphorylating the pore-forming α<sub>1C</sub> subunit or an auxiliary protein (<xref rid="bib1" ref-type="bibr">Anderson et al., 1994</xref>)?</p><p>Tentative answers to these questions can be put forward in the context of a working hypothesis that emerges from our findings on L-type channel–CaMKII interactions (<xref rid="fig8" ref-type="fig">Fig. 8</xref>). In a quiescent excitable cell, CaMKII is free in the cytoplasm (<xref rid="fig8" ref-type="fig">Fig. 8</xref>, bottom left) inasmuch as the inactive form of the kinase did not significantly interact with any of the cytoplasmic regions of α<sub>1C</sub>. After an initial Ca<sup>2+</sup> entry, recruitment to the channel takes place in an activity-dependent manner. CaM binding to soluble CaMKII targets the kinase to certain intracellular domains of α<sub>1C</sub>, and if the depolarization frequency suffices to produce CaMKII autophosphorylation on Thr<sup>286</sup>, the resulting displacement of the kinase's autoregulatory domain exposes a potent anchoring site for the α<sub>1C</sub> COOH terminus (<xref rid="fig8" ref-type="fig">Fig. 8</xref>, bottom middle). Observations that autophosphorylated CaMKII is concentrated at the myocyte sarcolemma (<xref rid="bib68" ref-type="bibr">Xiao et al., 1994</xref>; <xref rid="bib65" ref-type="bibr">Vinogradova et al., 2000</xref>) can be explained at least in part by a direct interaction of the kinase with α<sub>1C</sub>. Moreover, the requirement for a local Ca<sup>2+</sup> signal to trigger CDF (<xref rid="bib30" ref-type="bibr">Hryshko and Bers, 1990</xref>; <xref rid="bib65" ref-type="bibr">Vinogradova et al., 2000</xref>) would arise if the necessary phosphorylation could only be achieved by a tethered kinase that is modulated by CaM molecules in the immediate vicinity of the channel-anchored CaMKII.</p><fig id="fig8" position="float"><label>Figure 8.</label><caption><p>
<bold>Proposed mechanism of CaMKII binding to α<sub>1C</sub> to form a local and dedicated Ca<sup>2</sup></bold>
<sup><bold>+</bold></sup>
<bold>spike integrator for CDF.</bold> A catalytic core and autoregulatory domain for a prototypical CaMKII inactive subunit is shown on the bottom left (inactive is indicated by green). Ca<sup>2+</sup>/CaM activation and Thr<sup>286</sup> autophosphorylation displace the CaMKII autoregulatory domain within the catalytic lobe to activate the subunit (yellow) and to expose an α<sub>1C</sub> tethering site. The CaMKII holoenzyme remains bound to the α<sub>1C</sub> COOH terminus even after removal of the Ca<sup>2+</sup>/CaM stimulus, and CaMKII dephosphorylation produces an inactive subunit. CaMKII may remain tethered to other cytoplasmic domains of α<sub>1C</sub> as well. High depolarization frequencies would produce a threshold level of activated/autophosphorylated CaMKII subunits that increase the <italic>P</italic>
<sub>o</sub> of the channel via phosphorylation of the NH<sub>2</sub> and/or COOH termini (top left). At low depolarization frequencies and under the influence of phosphatase action, CaMKII activation would not be produced, favoring a low <italic>P</italic>
<sub>o</sub> for α<sub>1C</sub> (top right).</p></caption><graphic xlink:href="200505155f8"/></fig><p>Once established, this interaction may persist even after Ca<sup>2+</sup> is lowered and the kinase is completely dephosphorylated (<xref rid="fig7" ref-type="fig">Fig. 7</xref> D), so that CaMKII remains tightly tethered to the channel as long as the cell is intermittently active (<xref rid="fig8" ref-type="fig">Fig. 8</xref>, bottom right). This scenario capitalizes on the dodecameric structure of the CaMKII holoenzyme (<xref rid="bib37" ref-type="bibr">Kolodziej et al., 2000</xref>) by using one or more kinase subunits for the purpose of subcellular localization (<xref rid="fig8" ref-type="fig">Fig. 8</xref>, top left). The existence of multiple CaMKII interaction sites on α<sub>1C</sub> (<xref rid="fig3" ref-type="fig">Fig. 3</xref>) may serve to couple the channel and the kinase more tightly and/or orient the large, dodecameric kinase for efficient phosphorylation. The securing of CaMKII in close proximity to key substrate sites on intracellular loops of the channel protein produces a high rate of channel phosphorylation and promotes a pattern of gating with high <italic>P</italic>
<sub>o</sub> (mode 2; <xref rid="bib18" ref-type="bibr">Dzhura et al., 2000</xref>). Lowering of the frequency of Ca<sup>2+</sup> influx reduces kinase activation and allows phosphatases to prevail in dephosphorylating both the channel and its associated CaMKII, driving the channel into a low <italic>P</italic>
<sub>o</sub> gating mode (<xref rid="fig8" ref-type="fig">Fig. 8</xref>, top right). Because the resident CaMKII can be fully dephosphorylated while remaining associated with the channel, its modulatory activity can be graded over the widest possible working range. By virtue of its position, the anchored kinase has a tremendous kinetic advantage over cytosolic CaMKII molecules and essentially monopolizes the modulatory function. Accordingly, a mutation in α<sub>1C</sub> that rendered the cytoplasmic tail unable to bind CaMKII completely abolished CDF (<xref rid="fig5" ref-type="fig">Fig. 5</xref>). Thus, tethering of CaMKII to the COOH terminus of the channel is critical for making it competent for CDF. The combined channel–kinase complex represents a dedicated frequency detector that responds specifically to local Ca<sup>2+</sup> signaling.</p><p>Looking beyond Ca<sup>2+</sup> channels in surface membranes, Ca<sup>2+</sup> sequestration into intracellular Ca<sup>2+</sup> stores undergoes a frequency-dependent acceleration in myocardial cells, which is also critically dependent on CaMKII (<xref rid="bib15" ref-type="bibr">DeSantiago et al., 2002</xref>). It remains unclear whether this action of CaMKII depends on activity-dependent targeting and whether frequency-dependent modulation is a common feature of Ca<sup>2+</sup> signaling proteins (<xref rid="bib45" ref-type="bibr">Maier and Bers, 2002</xref>).</p></sec><sec><title>Comparisons with L-type channel modulation by other kinases</title><p>The tethering of CaMKII to α<sub>1C</sub> adds some unique elements to the repertoire of mechanisms used by signaling molecules to link stimulus to cellular response. The L-type channel–CaMKII interaction takes advantage of the multimeric CaMKII holoenzyme, using one or a limited number of its 12 catalytic subunits for anchoring and therefore circumventing the use of auxiliary proteins such as AKAPs or receptors for activated protein kinase C, which tether PKA or PKC, respectively (<xref rid="bib9" ref-type="bibr">Bunemann et al., 1999</xref>; <xref rid="bib64" ref-type="bibr">Tavalin et al., 1999</xref>; <xref rid="bib59" ref-type="bibr">Schechtman and Mochly-Rosen, 2001</xref>; <xref rid="bib17" ref-type="bibr">Dorn and Mochly-Rosen, 2002</xref>). Another distinction lies in the persistent tethering of CaMKII and its catalytic domains to α<sub>1C</sub>. The spatial zone of catalytic activity is delimited by the distance from site of anchored subunit to most distant subunit of that holoenzyme. Dissociation of the PKA R<sub>2</sub>C<sub>2</sub> complex from AKAPs leads to the immediate loss of catalytic localization once the C subunits are liberated and thereby activated over a much larger spatial volume. This mechanism is ideal for enabling catalytic subunits to diffuse from the site of activation to the nucleus (<xref rid="bib27" ref-type="bibr">Harootunian et al., 1993</xref>) and is acceptable if β-adrenergic potentiation of L-type Ca<sup>2+</sup> currents (<xref rid="bib23" ref-type="bibr">Gao et al., 1997</xref>; <xref rid="bib33" ref-type="bibr">Hulme et al., 2002</xref>) requires rapid responsiveness but only on infrequent occasions. The persistent tethering of the CaMKII holoenzyme might be better suited for continuous operation as an integrator of L-type Ca<sup>2+</sup> channel activity, endowed with briskly reversible Ca<sup>2+</sup> responsiveness and dedicated to a limited number of channels.</p></sec><sec><title>Similarities and contrasts with CaMKII–NMDAR interactions</title><p>Like L-type channels, NMDARs are predominant Ca<sup>2+</sup> entry pathways in neurons for triggering synaptic plasticity and signaling to the nucleus, and CaMKII is tethered to the NR1 and NR2B subunits of the NMDAR, so our experiments provide interesting points of comparison with previous work showing the direct binding of CaMKII to the NR2B and NR1 subunits of NMDARs (<xref rid="bib62" ref-type="bibr">Strack and Colbran, 1998</xref>; <xref rid="bib41" ref-type="bibr">Leonard et al., 1999</xref>, <xref rid="bib42" ref-type="bibr">2002</xref>; <xref rid="bib63" ref-type="bibr">Strack et al., 2000</xref>; <xref rid="bib2" ref-type="bibr">Bayer et al., 2001</xref>). There are telling similarities between NMDAR subunits and α<sub>1C</sub> as targets for CaMKII binding. First, completely inactive CaMKII will not initiate binding to any of these subunits. Second, in both NR2B and α<sub>1C</sub>, a COOH-terminal domain of the membrane protein competes with the autoregulatory domain of CaMKII for binding to the kinase, as shown by peptide competition (<xref rid="fig6" ref-type="fig">Fig. 6</xref> B; <xref rid="bib63" ref-type="bibr">Strack et al., 2000</xref>). This similarity was highlighted by the finding that a peptide based on the CaMKII binding site on NR2B prevented the kinase from interacting with the α<sub>1C</sub> COOH-terminal tail (<xref rid="fig6" ref-type="fig">Fig. 6</xref> B). Third, in both NR1 and α<sub>1C</sub>, the site of CaMKII binding lies close to a site for CaM binding. In the C0 domain of NR1, the amino acids most critical for CaMKII binding lie three residues NH<sub>2</sub>-terminal to those most important for CaM binding (<xref rid="bib42" ref-type="bibr">Leonard et al., 2002</xref>). Likewise, the α<sub>1C</sub> sequence implicated in the CaMKII interaction (Mut6) lies between stretches of amino acids, among them the IQ motif, that are critical for CaM tethering and effector action (<xref rid="bib54" ref-type="bibr">Peterson et al., 1999</xref>; <xref rid="bib71" ref-type="bibr">Zühlke et al., 1999</xref>, <xref rid="bib72" ref-type="bibr">2000</xref>; <xref rid="bib53" ref-type="bibr">Pate et al., 2000</xref>; <xref rid="bib56" ref-type="bibr">Romanin et al., 2000</xref>; <xref rid="bib55" ref-type="bibr">Pitt et al., 2001</xref>; <xref rid="bib20" ref-type="bibr">Erickson et al., 2003</xref>; <xref rid="bib35" ref-type="bibr">Kim et al., 2004</xref>). Further studies will be needed to understand how the activity of the anchored CaMKII may be integrated with the Ca<sup>2+</sup>-sensing properties of the CaM–IQ domain complex for regulation of L-type channel gating and for downstream signaling to nuclear cAMP response element–binding protein (<xref rid="bib16" ref-type="bibr">Dolmetsch et al., 2001</xref>).</p><p>There are also critical functional differences between α<sub>1C</sub> and NR2B in their interaction with CaMKII. Although the COOH-terminal tails of α<sub>1C</sub> and NR2B use overlapping sites on CaMKII for binding, the two channels exhibit significant differences in kinase activation state requirements and in consequences of tethering. The NR2B COOH terminus displays a high-affinity interaction with CaMKII that merely requires Ca<sup>2+</sup>/CaM activation of CaMKII, not autophosphorylation (<xref rid="bib2" ref-type="bibr">Bayer et al., 2001</xref>). In contrast, the COOH terminus of α<sub>1C</sub> only binds to autophosphorylated CaMKII (<xref rid="fig3" ref-type="fig">Fig. 3</xref>). Binding of CaMKII to NR2B alters kinase function, causing maintained kinase activity even in the absence of Ca<sup>2+</sup>/CaM or autophosphorylation. This is not the case for CaMKII binding to α<sub>1C</sub>; our experiments show that interaction with the α<sub>1C</sub> COOH terminus does not circumvent the autoinhibitory function of the bound kinase. The contrasting properties might arise from substantial differences in the respective COOH-terminal sequences of α<sub>1C</sub> and NR2B (<xref rid="fig6" ref-type="fig">Fig. 6</xref> C) and might offer specific advantages appropriate to the different roles of the two channels. Establishment of sustained CaMKII activity after transient NMDAR signaling seems perfectly appropriate as a means of supporting enduring effects, e.g., long-term potentiation and long-term depression (<xref rid="bib43" ref-type="bibr">Lisman et al., 2002</xref>). On the other hand, CDF of L-type channels would suffer a significant loss of dynamic range if the α<sub>1C</sub> COOH-terminal interaction with CaMKII were to cause constitutive kinase activity. The retention of dependence on Ca<sup>2+</sup>/CaM for enzymatic activity is well suited for the operation of CaMKII as a built-in integrator of the frequency of prior Ca<sup>2+</sup> signaling (<xref rid="bib31" ref-type="bibr">Hudmon and Schulman, 2002a</xref>; <xref rid="bib45" ref-type="bibr">Maier and Bers, 2002</xref>).</p></sec></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>Oocyte recordings</title><p>The plasmid encoding the rabbit cardiac α<sub>1C</sub> subunit used for expression in <italic>X. laevis</italic> oocytes, pCARDHE, was a gift of W. Sather (University of Colorado, Denver, CO). In vitro transcription and microinjection into <italic>X. laevis</italic> oocytes (provided by J. Riley and S. Siegelbaum, Columbia University, New York, NY) of α<sub>1C</sub>, the auxiliary Ca<sup>2+</sup> channel subunits β<sub>1</sub> and α<sub>2</sub>δ, were performed as previously described (<xref rid="bib72" ref-type="bibr">Zühlke et al., 2000</xref>). Before recording whole cell <italic>I</italic>
<sub>Ba</sub> or <italic>I</italic>
<sub>Ca</sub>, oocytes were injected with 25–50 nl of 100 mM BAPTA solution, pH 7.4, to minimize contaminating Ca<sup>2+</sup>-activated Cl<sup>−</sup> currents. <italic>I</italic>
<sub>Ba</sub> and <italic>I</italic>
<sub>Ca</sub> recordings were performed essentially as described previously (<xref rid="bib72" ref-type="bibr">Zühlke et al., 2000</xref>) with a standard two-electrode voltage clamp configuration using an oocyte clamp amplifier (OC-725C; Warner Instrument Corp.) connected through a Digidata 3122A A/D interface (Axon Instruments, Inc.) to a personal computer. <italic>I</italic>
<sub>Ba</sub> and <italic>I</italic>
<sub>Ca</sub> were recorded in the same oocyte. Ionic currents were filtered at 1 kHz by an integral 4-pole Bessel filter, sampled at 10 kHz, and analyzed with Clampfit 8.1.</p></sec><sec><title>GST fusion proteins</title><p>PCR fragments corresponding to the a1C (available from Genbank/EMBL/DDBJ under accession no. <ext-link ext-link-type="gen" xlink:href="X15539">X15539</ext-link>) NH2 terminus (aa 1–154), I-II intracellular loop (aa 435–554), II-III intracellular loop (aa 784–931), III-IV intracellular loop (aa 1197–1250), and two COOH-terminal fragments (aa 1581–1690 and 1669–2171) were cloned into pGEX-4T-1, and GST fusion proteins were generated. The plasmids encoding the COOH-terminal fragments CT5 (aa 1507–1622), CT12 (aa 1509–1905), and CT23 (aa 1622–1905) were provided by M. Hosey (Northwestern University, Evanston, IL).</p></sec><sec><title>Peptides</title><p>Peptides spanning α<sub>1C</sub> residues 1581–1690 have previously been described (<xref rid="bib55" ref-type="bibr">Pitt et al., 2001</xref>). The <italic>N</italic>-methyl-<sc>d</sc>-aspartate-<sc>l</sc> peptide (<xref rid="bib2" ref-type="bibr">Bayer et al., 2001</xref>) and peptides AC-2 (<xref rid="bib26" ref-type="bibr">Hanson et al., 1989</xref>), AC-3i (<xref rid="bib8" ref-type="bibr">Braun and Schulman, 1995</xref>), and AC-3c (<xref rid="bib8" ref-type="bibr">Braun and Schulman, 1995</xref>) have been described elsewhere.</p></sec><sec><title>Immunoprecipitation</title><p>Rat cardiac sarcolemmal membranes were provided by S.O. Marx (Columbia University, New York, NY). Immunoprecipitation was performed with either an anti-α<sub>1C</sub> (Alomone) or control IgG in 150 mM NaCl, 50 mM Tris, pH 8.0, 1% Triton, and Complete protease inhibitor cocktail (Roche). After SDS-PAGE, calmodulin overlay was performed with biotin-conjugated calmodulin (STI Signal Transduction) and detected with Vectastain ABC kit (Vector Laboratories). HEK 293 cells were transfected with α<sub>1C</sub>, α<sub>2</sub>δ, β2, and GFP-CaMKII using Lipofectamine 2000 (Invitrogen) as instructed by the manufacturer. After 48 h, they were washed in ice-cold PBS and then lysed in 150 mM NaCl, 50 mM Tris, pH 8.0, 1% Triton, and Complete protease inhibitor cocktail, and immunoprecipitation was performed with the anti-α<sub>1C</sub> antibody (Alamone). After SDS-PAGE, immunoblotting was performed with an anti-GFP antibody (Covance).</p></sec><sec><title>Expression and purification of CaMKII</title><p>α-CaMKII was expressed and purified essentially as described previously (<xref rid="bib6" ref-type="bibr">Bradshaw et al., 2002</xref>). Additional CaMKII isoforms were generated by transient expression in HEK 293 cells (Srα plasmid containing the α, β, δ<sub>A</sub>, δ<sub>C</sub>, or γ<sub>B</sub> isoforms). After 72 h, cells were lysed in 10 mM Tris/5% Betaine/150 mM sodium perchlorate, pH 7.5, by brief sonication. Cell lysates were centrifuged for 30 min at 14,000 <italic>g</italic> at 4°C, and the supernatants were aliquoted, snap frozen, and stored at –80°C.</p></sec><sec><title>GST binding assay</title><p>The binding reactions were accomplished in Tris-binding buffer (50 mM Tris, 150 mM NaCl, 0.1% T-20, pH 7.4, and 0.1% BSA) containing 20 nM purified CaMKII. The total protein from the HEK 293 cell lysates added to each binding reaction ranged from 9 to 22 μg, as determined by normalizing for the amount of CaMKII activity (<xref rid="bib61" ref-type="bibr">Singla et al., 2001</xref>). Preautophosphorylayion of CaMKII (purified and lysate) was performed on ice for 5 min in Tris-binding buffer plus 1 mM CaCl<sub>2</sub>, 5 μM CaM, 1 mM ATP, and 5 mM MgCl<sub>2</sub> to restrict the sites of autophosphorylation to primarily Thr<sup>286</sup> (<xref rid="bib38" ref-type="bibr">Lai et al., 1987</xref>; <xref rid="bib44" ref-type="bibr">Lou and Schulman, 1989</xref>; <xref rid="bib34" ref-type="bibr">Ikeda et al., 1991</xref>). Final concentration of these components in the binding reaction (1:40) was 0.025 mM CaCl<sub>2</sub>, 0.125 μM CaM, 0.025 mM ATP, and 0.125 mM MgCl<sub>2</sub>. The binding reaction was rocked for 1 h at 4°C, and the beads were extensively washed in Tris-binding buffer (2–3 times for 5 min each). CaMKII binding was quantified using densiotometric measurement of band intensity using 1D Image Analysis Software (Eastman Kodak Co.). Multiple exposure times, as well as a standard curve generated by dilution analysis, ensured linearity in the chemiluminescence intensity. One-way analysis of variance was performed, and Dunnett's test was used to identify specific pair-wise differences between the means. Comparison analyses were conducted using SPSS Version 10.1.3 (SPSS, Inc.).</p></sec><sec><title>Calmodulin binding assay</title><p>The bound GST proteins–sepharose complex was prepared as described in the previous section. Purified CaM (<xref rid="bib61" ref-type="bibr">Singla et al., 2001</xref>) was applied in the presence of 1 mM CaCl<sub>2</sub> for 1 h before multiple washes of Tris-binding buffer plus 1 mM CaCl<sub>2</sub>. Immunoblotting was performed as described previously (<xref rid="bib55" ref-type="bibr">Pitt et al., 2001</xref>).</p></sec><sec><title>CaMKII phosphorylation of α<sub>1C</sub>
</title><p>Purified α-CaMKII was incubated with bound GST fusion proteins or immunoprecipitated material bound to PKA in the presence of Ca<sup>2+</sup>/CaM (2 mM/10 μM) and Mg<sup>2+</sup>/ATP (5 mM/50 μM ATP) plus 10–50 μCi ATP<sup>32</sup> for 15 min at RT. For the GST proteins, CaMKII was activated before exposure to the substrate reaction on ice (as described in GST binding assay) to produce an autophosphorylated enzyme. After the phosphorylation, the beads were washed extensively in PBS (plus 5 mM EDTA) and 2× SDS-PAGE sample buffer was added and SDS-PAGE was performed. The gels were Coomassie stained and exhaustively destained. The gels were dried down, and P<sup>32</sup>-labeled proteins were detected using autoradiography.</p></sec><sec><title>CaMKII dephosphorylation using PP1</title><p>CaMKII was dephosphorylated using a Hisx6-tagged PP1 catalytic subunit construct (provided by A. Nairn, Yale University, New Haven, CT) purified by Ni-NTA affinity chromatography (provided by M. Bradshaw, Stanford University, Stanford, CA).</p></sec></sec></body><back><ack><p>The authors are grateful to Ben Barres and Michael Bradshaw for helpful discussion and comments on this manuscript.</p><p>This work was supported by grants from the National Institutes of Health to H. Schulman, R.W. Tsien, and G.S. Pitt; a grant from the Irma T. Hirschl Monique Weill-Caulier Trust to G.S. Pitt; and an award from the American Heart Association to A. Hudmon.</p></ack><ref-list><ref id="bib1"><mixed-citation publication-type="journal">Anderson, M., A. Braun, H. Schulman, and B. Premack. <year>1994</year>. Multifunctional Ca<sup>2+</sup>/calmodulin-dependent protein kinase mediates Ca<sup>2+</sup>-induced enhancement of the L-type Ca<sup>2+</sup> current in rabbit ventricular myocytes. <source>Circ. Res.</source>
<volume>75</volume>:<fpage>854</fpage>–861.
<pub-id pub-id-type="pmid">7923631</pub-id></mixed-citation></ref><ref id="bib2"><mixed-citation publication-type="journal">Bayer, K.U., P. De Koninck, A.S. Leonard, J.W. Hell, and H. Schulman. <year>2001</year>. Interaction with the NMDA receptor locks CaMKII in an active conformation. <source>Nature.</source>
<volume>411</volume>:<fpage>801</fpage>–805.
<pub-id pub-id-type="pmid">11459059</pub-id></mixed-citation></ref><ref id="bib3"><mixed-citation publication-type="journal">Bayer, K.U., P. De Koninck, and H. Schulman. <year>2002</year>. Alternative splicing modulates the frequency-dependent response of CaMKII to Ca<sup>2+</sup> oscillations. <source>EMBO J.</source>
<volume>21</volume>:<fpage>3590</fpage>–3597.
<pub-id pub-id-type="pmid">12110572</pub-id></mixed-citation></ref><ref id="bib4"><mixed-citation publication-type="journal">Bence-Hanulec, K.K., J. Marshall, and L.A. Blair. <year>2000</year>. Potentiation of neuronal L calcium channels by IGF-1 requires phosphorylation of the α<sub>1</sub> subunit on a specific tyrosine residue. <source>Neuron.</source>
<volume>27</volume>:<fpage>121</fpage>–131.
<pub-id pub-id-type="pmid">10939336</pub-id></mixed-citation></ref><ref id="bib5"><mixed-citation publication-type="journal">Bradley, J., and S. Finkbeiner. <year>2002</year>. An evaluation of specificity in activity-dependent gene expression in neurons. <source>Prog. Neurobiol.</source>
<volume>67</volume>:<fpage>469</fpage>–477.
<pub-id pub-id-type="pmid">12385865</pub-id></mixed-citation></ref><ref id="bib6"><mixed-citation publication-type="journal">Bradshaw, J.M., A. Hudmon, and H. Schulman. <year>2002</year>. Chemical quenched flow kinetic studies indicate an intraholoenzyme autophosphorylation mechanism for Ca<sup>2+</sup>/calmodulin-dependent protein kinase II. <source>J. Biol. Chem.</source>
<volume>277</volume>:<fpage>20991</fpage>–20998.
<pub-id pub-id-type="pmid">11925447</pub-id></mixed-citation></ref><ref id="bib7"><mixed-citation publication-type="journal">Bradshaw, J.M., Y. Kubota, T. Meyer, and H. Schulman. <year>2003</year>. An ultrasensitive Ca<sup>2+</sup>/calmodulin-dependent protein kinase II-protein phosphatase 1 switch facilitates specificity in postsynaptic calcium signaling. <source>Proc. Natl. Acad. Sci. USA.</source>
<volume>100</volume>:<fpage>10512</fpage>–10517.
<pub-id pub-id-type="pmid">12928489</pub-id></mixed-citation></ref><ref id="bib8"><mixed-citation publication-type="journal">Braun, A.P., and H. Schulman. <year>1995</year>. A non-selective cation current activated via the multifunctional Ca<sup>2+</sup>-calmodulin-dependent protein kinase in human epithelial cells. <source>J. Physiol.</source>
<volume>488</volume>:<fpage>37</fpage>–55.
<pub-id pub-id-type="pmid">8568664</pub-id></mixed-citation></ref><ref id="bib9"><mixed-citation publication-type="journal">Bunemann, M., B.L. Gerhardstein, T. Gao, and M.M. Hosey. <year>1999</year>. Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the β<sub>2</sub> subunit. <source>J. Biol. Chem.</source>
<volume>274</volume>:<fpage>33851</fpage>–33854.
<pub-id pub-id-type="pmid">10567342</pub-id></mixed-citation></ref><ref id="bib10"><mixed-citation publication-type="journal">Cuttle, M.F., T. Tsujimoto, I.D. Forsythe, and T. Takahashi. <year>1998</year>. Facilitation of the presynaptic calcium current at an auditory synapse in rat brainstem. <source>J. Physiol.</source>
<volume>512</volume>:<fpage>723</fpage>–729.
<pub-id pub-id-type="pmid">9769416</pub-id></mixed-citation></ref><ref id="bib11"><mixed-citation publication-type="journal">De Jongh, K.S., B.J. Murphy, A.A. Colvin, J.W. Hell, M. Takahashi, and W.A. Catterall. <year>1996</year>. Specific phosphorylation of a site in the full-length form of the α1 subunit of the cardiac L-type calcium channel by adenosine 3′,5′-cyclic monophosphate-dependent protein kinase. <source>Biochemistry.</source>
<volume>35</volume>:<fpage>10392</fpage>–10402.
<pub-id pub-id-type="pmid">8756695</pub-id></mixed-citation></ref><ref id="bib12"><mixed-citation publication-type="journal">De Koninck, P., and H. Schulman. <year>1998</year>. Sensitivity of CaM kinase II to the frequency of Ca<sup>2+</sup> oscillations. <source>Science.</source>
<volume>279</volume>:<fpage>227</fpage>–230.
<pub-id pub-id-type="pmid">9422695</pub-id></mixed-citation></ref><ref id="bib13"><mixed-citation publication-type="journal">Deisseroth, K., P.G. Mermelstein, H. Xia, and R.W. Tsien. <year>2003</year>. Signaling from synapse to nucleus: the logic behind the mechanisms. <source>Curr. Opin. Neurobiol.</source>
<volume>13</volume>:<fpage>354</fpage>–365.
<pub-id pub-id-type="pmid">12850221</pub-id></mixed-citation></ref><ref id="bib14"><mixed-citation publication-type="journal">DeMaria, C.D., T.W. Soong, B.A. Alseikhan, R.S. Alvania, and D.T. Yue. <year>2001</year>. Calmodulin bifurcates the local Ca<sup>2+</sup> signal that modulates P/Q-type Ca<sup>2+</sup> channels. <source>Nature.</source>
<volume>411</volume>:<fpage>484</fpage>–489.
<pub-id pub-id-type="pmid">11373682</pub-id></mixed-citation></ref><ref id="bib15"><mixed-citation publication-type="journal">DeSantiago, J., L.S. Maier, and D.M. Bers. <year>2002</year>. Frequency-dependent acceleration of relaxation in the heart depends on CaMKII, but not phospholamban. <source>J. Mol. Cell. Cardiol.</source>
<volume>34</volume>:<fpage>975</fpage>–984.
<pub-id pub-id-type="pmid">12234767</pub-id></mixed-citation></ref><ref id="bib16"><mixed-citation publication-type="journal">Dolmetsch, R.E., U. Pajvani, K. Fife, J.M. Spotts, and M.E. Greenberg. <year>2001</year>. Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. <source>Science.</source>
<volume>294</volume>:<fpage>333</fpage>–339.
<pub-id pub-id-type="pmid">11598293</pub-id></mixed-citation></ref><ref id="bib17"><mixed-citation publication-type="journal">Dorn, G.W., II, and D. Mochly-Rosen. <year>2002</year>. Intracellular transport mechanisms of signal transducers. <source>Annu. Rev. Physiol.</source>
<volume>64</volume>:<fpage>407</fpage>–429.
<pub-id pub-id-type="pmid">11826274</pub-id></mixed-citation></ref><ref id="bib18"><mixed-citation publication-type="journal">Dzhura, I., Y. Wu, R.J. Colbran, J.R. Balser, and M.E. Anderson. <year>2000</year>. Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels. <source>Nat. Cell Biol.</source>
<volume>2</volume>:<fpage>173</fpage>–177.
<pub-id pub-id-type="pmid">10707089</pub-id></mixed-citation></ref><ref id="bib19"><mixed-citation publication-type="journal">Edman, C.F., and H. Schulman. <year>1994</year>. Identification and characterization of δ<sub>B</sub>-CaM kinase and δ<sub>C</sub>-CaM kinase from rat heart, two new multifunctional Ca<sup>2+</sup>/calmodulin-dependent protein kinase isoforms. <source>Biochim. Biophys. Acta.</source>
<volume>1221</volume>:<fpage>89</fpage>–101.
<pub-id pub-id-type="pmid">8130281</pub-id></mixed-citation></ref><ref id="bib20"><mixed-citation publication-type="journal">Erickson, M.G., H. Liang, M.X. Mori, and D.T. Yue. <year>2003</year>. FRET two-hybrid mapping reveals function and location of L-type Ca<sup>2+</sup> channel CaM preassociation. <source>Neuron.</source>
<volume>39</volume>:<fpage>97</fpage>–107.
<pub-id pub-id-type="pmid">12848935</pub-id></mixed-citation></ref><ref id="bib21"><mixed-citation publication-type="journal">Eshete, F., and R.D. Fields. <year>2001</year>. Spike frequency decoding and autonomous activation of Ca<sup>2+</sup>-calmodulin-dependent protein kinase II in dorsal root ganglion neurons. <source>J. Neurosci.</source>
<volume>21</volume>:<fpage>6694</fpage>–6705.
<pub-id pub-id-type="pmid">11517259</pub-id></mixed-citation></ref><ref id="bib22"><mixed-citation publication-type="journal">Feldman, M.D., J.D. Alderman, J.M. Aroesty, H.D. Royal, J.J. Ferguson, R.M. Owen, W. Grossman, and R.G. McKay. <year>1988</year>. Depression of systolic and diastolic myocardial reserve during atrial pacing tachycardia in patients with dilated cardiomyopathy. <source>J. Clin. Invest.</source>
<volume>82</volume>:<fpage>1661</fpage>–1669.
<pub-id pub-id-type="pmid">3183060</pub-id></mixed-citation></ref><ref id="bib23"><mixed-citation publication-type="journal">Gao, T., A. Yatani, M.L. Dell'Acqua, H. Sako, S.A. Green, N. Dascal, J.D. Scott, and M.M. Hosey. <year>1997</year>. cAMP-dependent regulation of cardiac L-type Ca<sup>2+</sup> channels requires membrane targeting of PKA and phosphorylation of channel subunits. <source>Neuron.</source>
<volume>19</volume>:<fpage>185</fpage>–196.
<pub-id pub-id-type="pmid">9247274</pub-id></mixed-citation></ref><ref id="bib24"><mixed-citation publication-type="journal">Gao, T., A.E. Cuadra, H. Ma, M. Bunemann, B.L. Gerhardstein, T. Cheng, R.T. Eick, and M.M. Hosey. <year>2001</year>. C-terminal fragments of the α<sub>1C</sub> (Ca<sub>V</sub>1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated α<sub>1C</sub> subunits. <source>J. Biol. Chem.</source>
<volume>276</volume>:<fpage>21089</fpage>–21097.
<pub-id pub-id-type="pmid">11274161</pub-id></mixed-citation></ref><ref id="bib25"><mixed-citation publication-type="journal">Glenney, J.R., Jr., and K. Weber. <year>1983</year>. Detection of calmodulin-binding polypeptides separated in SDS-polyacrylamide gels by a sensitive [<sup>125</sup>I]calmodulin gel overlay assay. <source>Methods Enzymol.</source>
<volume>102</volume>:<fpage>204</fpage>–210.
<pub-id pub-id-type="pmid">6316075</pub-id></mixed-citation></ref><ref id="bib26"><mixed-citation publication-type="journal">Hanson, P.I., M.S. Kapiloff, L.L. Lou, M.G. Rosenfeld, and H. Schulman. <year>1989</year>. Expression of a multifunctional Ca<sup>2+</sup>/calmodulin-dependent protein kinase and mutational analysis of its autoregulation. <source>Neuron.</source>
<volume>3</volume>:<fpage>59</fpage>–70.
<pub-id pub-id-type="pmid">2619995</pub-id></mixed-citation></ref><ref id="bib27"><mixed-citation publication-type="journal">Harootunian, A.T., S.R. Adams, W. Wen, J.L. Meinkoth, S.S. Taylor, and R.Y. Tsien. <year>1993</year>. Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion. <source>Mol. Biol. Cell.</source>
<volume>4</volume>:<fpage>993</fpage>–1002.
<pub-id pub-id-type="pmid">8298196</pub-id></mixed-citation></ref><ref id="bib28"><mixed-citation publication-type="journal">Hasenfuss, G., C. Holubarsch, H.P. Hermann, K. Astheimer, B. Pieske, and H. Just. <year>1994</year>. Influence of the force-frequency relationship on haemodynamics and left ventricular function in patients with non-failing hearts and in patients with dilated cardiomyopathy. <source>Eur. Heart J.</source>
<volume>15</volume>:<fpage>164</fpage>–170.
<pub-id pub-id-type="pmid">8005115</pub-id></mixed-citation></ref><ref id="bib29"><mixed-citation publication-type="journal">Hell, J.W., C.T. Yokoyama, L.J. Breeze, C. Chavkin, and W.A. Catterall. <year>1995</year>. Phosphorylation of presynaptic and postsynaptic calcium channels by cAMP-dependent protein kinase in hippocampal neurons. <source>EMBO J.</source>
<volume>14</volume>:<fpage>3036</fpage>–3044.
<pub-id pub-id-type="pmid">7621818</pub-id></mixed-citation></ref><ref id="bib30"><mixed-citation publication-type="journal">Hryshko, L.V., and D.M. Bers. <year>1990</year>. Ca current facilitation during postrest recovery depends on Ca entry. <source>Am. J. Physiol.</source>
<volume>259</volume>:<fpage>H951</fpage>–H961.
<pub-id pub-id-type="pmid">2168683</pub-id></mixed-citation></ref><ref id="bib31"><mixed-citation publication-type="journal">Hudmon, A., and H. Schulman. <year>2002</year>a. Neuronal Ca<sup>2+</sup>/calmodulin-dependent protein kinase II: the role of structure and autoregulation in cellular function. <source>Annu. Rev. Biochem.</source>
<volume>71</volume>:<fpage>473</fpage>–510.
<pub-id pub-id-type="pmid">12045104</pub-id></mixed-citation></ref><ref id="bib32"><mixed-citation publication-type="journal">Hudmon, A., and H. Schulman. <year>2002</year>b. Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II. <source>Biochem. J.</source>
<volume>364</volume>:<fpage>593</fpage>–611.
<pub-id pub-id-type="pmid">11931644</pub-id></mixed-citation></ref><ref id="bib33"><mixed-citation publication-type="journal">Hulme, J.T., M. Ahn, S.D. Hauschka, T. Scheuer, and W.A. Catterall. <year>2002</year>. A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca<sup>2+</sup> channel and modulates its function. <source>J. Biol. Chem.</source>
<volume>277</volume>:<fpage>4079</fpage>–4087.
<pub-id pub-id-type="pmid">11733497</pub-id></mixed-citation></ref><ref id="bib34"><mixed-citation publication-type="journal">Ikeda, A., S. Okuno, and H. Fujisawa. <year>1991</year>. Studies on the generation of Ca<sup>2+</sup>/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme. <source>J. Biol. Chem.</source>
<volume>266</volume>:<fpage>11582</fpage>–11588.
<pub-id pub-id-type="pmid">1646810</pub-id></mixed-citation></ref><ref id="bib35"><mixed-citation publication-type="journal">Kim, J., S. Ghosh, D.A. Nunziato, and G.S. Pitt. <year>2004</year>. Isolation of the components controlling inactivation of voltage-gated Ca<sup>2+</sup> channels. <source>Neuron.</source>
<volume>41</volume>:<fpage>745</fpage>–754.
<pub-id pub-id-type="pmid">15003174</pub-id></mixed-citation></ref><ref id="bib36"><mixed-citation publication-type="journal">Koch-Weser, J., and J. Blinks. <year>1963</year>. The influence of the interval between beats on myocardial contractility. <source>Pharmacol. Rev.</source>
<volume>15</volume>:<fpage>601</fpage>–652.
<pub-id pub-id-type="pmid">14064358</pub-id></mixed-citation></ref><ref id="bib37"><mixed-citation publication-type="journal">Kolodziej, S.J., A. Hudmon, M.N. Waxham, and J.K. Stoops. <year>2000</year>. Three-dimensional reconstructions of calcium/calmodulin-dependent (CaM) kinase IIα and truncated CaM kinase IIα reveal a unique organization for its structural core and functional domains. <source>J. Biol. Chem.</source>
<volume>275</volume>:<fpage>14354</fpage>–14359.
<pub-id pub-id-type="pmid">10799516</pub-id></mixed-citation></ref><ref id="bib38"><mixed-citation publication-type="journal">Lai, Y., A.C. Nairn, F. Gorelick, and P. Greengard. <year>1987</year>. Ca<sup>2+</sup>/calmodulin-dependent protein kinase II: identification of autophosphorylation sites responsible for generation of Ca<sup>2+</sup>/calmodulin-independence. <source>Proc. Natl. Acad. Sci. USA.</source>
<volume>84</volume>:<fpage>5710</fpage>–5714.
<pub-id pub-id-type="pmid">3475699</pub-id></mixed-citation></ref><ref id="bib39"><mixed-citation publication-type="journal">Lee, A., S.T. Wong, D. Gallagher, B. Li, D.R. Storm, T. Scheuer, and W.A. Catterall. <year>1999</year>. Ca<sup>2+</sup>/calmodulin binds to and modulates P/Q-type calcium channels. <source>Nature.</source>
<volume>399</volume>:<fpage>155</fpage>–159.
<pub-id pub-id-type="pmid">10335845</pub-id></mixed-citation></ref><ref id="bib40"><mixed-citation publication-type="journal">Lee, K.S. <year>1987</year>. Potentiation of the calcium-channel currents of internally perfused mammalian heart cells by repetitive depolarization. <source>Proc. Natl. Acad. Sci. USA.</source>
<volume>84</volume>:<fpage>3941</fpage>–3945.
<pub-id pub-id-type="pmid">2438689</pub-id></mixed-citation></ref><ref id="bib41"><mixed-citation publication-type="journal">Leonard, A.S., I.A. Lim, D.E. Hemsworth, M.C. Horne, and J.W. Hell. <year>1999</year>. Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-D-aspartate receptor. <source>Proc. Natl. Acad. Sci. USA.</source>
<volume>96</volume>:<fpage>3239</fpage>–3244.
<pub-id pub-id-type="pmid">10077668</pub-id></mixed-citation></ref><ref id="bib42"><mixed-citation publication-type="journal">Leonard, A.S., K.-U. Bayer, M.A. Merrill, I.A. Lim, M.A. Shea, H. Schulman, and J.W. Hell. <year>2002</year>. Regulation of calcium/calmodulin-dependent protein kinase II docking to N-methyl-D-aspartate receptors by calcium/calmodulin and α-actinin. <source>J. Biol. Chem.</source>
<volume>277</volume>:<fpage>48441</fpage>–48448.
<pub-id pub-id-type="pmid">12379661</pub-id></mixed-citation></ref><ref id="bib43"><mixed-citation publication-type="journal">Lisman, J., H. Schulman, and H. Cline. <year>2002</year>. The molecular basis of CaMKII function in synaptic and behavioural memory. <source>Nat. Rev. Neurosci.</source>
<volume>3</volume>:<fpage>175</fpage>–190.
<pub-id pub-id-type="pmid">11994750</pub-id></mixed-citation></ref><ref id="bib44"><mixed-citation publication-type="journal">Lou, L.L., and H. Schulman. <year>1989</year>. Distinct autophosphorylation sites sequentially produce autonomy and inhibition of the multifunctional Ca<sup>2+</sup>/calmodulin-dependent protein kinase. <source>J. Neurosci.</source>
<volume>9</volume>:<fpage>2020</fpage>–2032.
<pub-id pub-id-type="pmid">2542484</pub-id></mixed-citation></ref><ref id="bib45"><mixed-citation publication-type="journal">Maier, L.S., and D.M. Bers. <year>2002</year>. Calcium, calmodulin, and calcium-calmodulin kinase II: heartbeat to heartbeat and beyond. <source>J. Mol. Cell. Cardiol.</source>
<volume>34</volume>:<fpage>919</fpage>–939.
<pub-id pub-id-type="pmid">12234763</pub-id></mixed-citation></ref><ref id="bib46"><mixed-citation publication-type="journal">Marban, E., and R.W. Tsien. <year>1982</year>. Enhancement of calcium current during digitalis inotropy in mammalian heart: positive feed-back regulation by intracellular calcium? <source>J. Physiol.</source>
<volume>329</volume>:<fpage>589</fpage>–614.
<pub-id pub-id-type="pmid">6292410</pub-id></mixed-citation></ref><ref id="bib47"><mixed-citation publication-type="journal">McCarron, J.G., J.G. McGeown, S. Reardon, M. Ikebe, F.S. Fay, and J.V. Walsh Jr. <year>1992</year>. Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II. <source>Nature.</source>
<volume>357</volume>:<fpage>74</fpage>–77.
<pub-id pub-id-type="pmid">1315424</pub-id></mixed-citation></ref><ref id="bib48"><mixed-citation publication-type="journal">McHugh, D., E.M. Sharp, T. Scheuer, and W.A. Catterall. <year>2000</year>. Inhibition of cardiac L-type calcium channels by protein kinase C phosphorylation of two sites in the N-terminal domain. <source>Proc. Natl. Acad. Sci. USA.</source>
<volume>97</volume>:<fpage>12334</fpage>–12338.
<pub-id pub-id-type="pmid">11035786</pub-id></mixed-citation></ref><ref id="bib49"><mixed-citation publication-type="journal">Meyer, T., P.I. Hanson, L. Stryer, and H. Schulman. <year>1992</year>. Calmodulin trapping by calcium-calmodulin-dependent protein kinase. <source>Science.</source>
<volume>256</volume>:<fpage>1199</fpage>–1202.
<pub-id pub-id-type="pmid">1317063</pub-id></mixed-citation></ref><ref id="bib50"><mixed-citation publication-type="journal">Miller, S.G., B.L. Patton, and M.B. Kennedy. <year>1988</year>. Sequences of autophosphorylation sites in neuronal type II CaM kinase that control Ca<sup>2+</sup>-independent activity. <source>Neuron.</source>
<volume>1</volume>:<fpage>593</fpage>–604.
<pub-id pub-id-type="pmid">2856100</pub-id></mixed-citation></ref><ref id="bib51"><mixed-citation publication-type="journal">Mulieri, L.A., G. Hasenfuss, B. Leavitt, P.D. Allen, and N.R. Alpert. <year>1992</year>. Altered myocardial force-frequency relation in human heart failure. <source>Circulation.</source>
<volume>85</volume>:<fpage>1743</fpage>–1750.
<pub-id pub-id-type="pmid">1572031</pub-id></mixed-citation></ref><ref id="bib52"><mixed-citation publication-type="journal">Noble, S., and Y. Shimoni. <year>1981</year>. The calcium and frequency dependence of the slow inward current ‘staircase’ in frog atrium. <source>J. Physiol.</source>
<volume>310</volume>:<fpage>57</fpage>–75.
<pub-id pub-id-type="pmid">6785423</pub-id></mixed-citation></ref><ref id="bib53"><mixed-citation publication-type="journal">Pate, P., J. Mochca-Morales, Y. Wu, J.Z. Zhang, G.G. Rodney, I.I. Serysheva, B.Y. Williams, M.E. Anderson, and S.L. Hamilton. <year>2000</year>. Determinants for calmodulin binding on voltage-dependent Ca<sup>2+</sup> channels. <source>J. Biol. Chem.</source>
<volume>275</volume>:<fpage>39786</fpage>–39792.
<pub-id pub-id-type="pmid">11005820</pub-id></mixed-citation></ref><ref id="bib54"><mixed-citation publication-type="journal">Peterson, B.Z., C.D. DeMaria, J.P. Adelman, and D.T. Yue. <year>1999</year>. Calmodulin is the Ca<sup>2+</sup> sensor for Ca<sup>2+</sup>-dependent inactivation of L-type calcium channels. <source>Neuron.</source>
<volume>22</volume>:<fpage>549</fpage>–558. (published erratum appears in <italic>Neuron.</italic> 1999. 22:following 893)
<pub-id pub-id-type="pmid">10197534</pub-id></mixed-citation></ref><ref id="bib55"><mixed-citation publication-type="journal">Pitt, G.S., R.D. Zuhlke, A. Hudmon, H. Schulman, H. Reuter, and R.W. Tsien. <year>2001</year>. Molecular basis of calmodulin tethering and Ca<sup>2+</sup>-dependent inactivation of L-type Ca<sup>2+</sup> channels. <source>J. Biol. Chem.</source>
<volume>276</volume>:<fpage>30794</fpage>–30802.
<pub-id pub-id-type="pmid">11408490</pub-id></mixed-citation></ref><ref id="bib56"><mixed-citation publication-type="journal">Romanin, C., R. Gamsjaeger, H. Kahr, D. Schaufler, O. Carlson, D.R. Abernethy, and N.M. Soldatov. <year>2000</year>. Ca<sup>2+</sup> sensors of L-type Ca<sup>2+</sup> channel. <source>FEBS Lett.</source>
<volume>487</volume>:<fpage>301</fpage>–306.
<pub-id pub-id-type="pmid">11150529</pub-id></mixed-citation></ref><ref id="bib57"><mixed-citation publication-type="journal">Ross, J., Jr., T. Miura, M. Kambayashi, G.P. Eising, and K.-H. Ryu. <year>1995</year>. Adrenergic control of the force-frequency relation. <source>Circulation.</source>
<volume>92</volume>:<fpage>2327</fpage>–2332.
<pub-id pub-id-type="pmid">7554218</pub-id></mixed-citation></ref><ref id="bib58"><mixed-citation publication-type="journal">Rotman, E.I., B.J. Murphy, and W.A. Catterall. <year>1995</year>. Sites of selective cAMP-dependent phosphorylation of the L-type calcium channel α<sub>1</sub> subunit from intact rabbit skeletal muscle myotubes. <source>J. Biol. Chem.</source>
<volume>270</volume>:<fpage>16371</fpage>–16377.
<pub-id pub-id-type="pmid">7608207</pub-id></mixed-citation></ref><ref id="bib59"><mixed-citation publication-type="journal">Schechtman, D., and D. Mochly-Rosen. <year>2001</year>. Adaptor proteins in protein kinase C-mediated signal transduction. <source>Oncogene.</source>
<volume>20</volume>:<fpage>6339</fpage>–6347.
<pub-id pub-id-type="pmid">11607837</pub-id></mixed-citation></ref><ref id="bib60"><mixed-citation publication-type="journal">Schouten, V.J., and M. Morad. <year>1989</year>. Regulation of Ca<sup>2+</sup> current in frog ventricular myocytes by the holding potential, c-AMP and frequency. <source>Pflugers Arch.</source>
<volume>415</volume>:<fpage>1</fpage>–11.
<pub-id pub-id-type="pmid">2560160</pub-id></mixed-citation></ref><ref id="bib61"><mixed-citation publication-type="journal">Singla, S.I., A. Hudmon, J.M. Goldberg, J.L. Smith, and H. Schulman. <year>2001</year>. Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II. <source>J. Biol. Chem.</source>
<volume>276</volume>:<fpage>29353</fpage>–29360.
<pub-id pub-id-type="pmid">11384969</pub-id></mixed-citation></ref><ref id="bib62"><mixed-citation publication-type="journal">Strack, S., and R.J. Colbran. <year>1998</year>. Autophosphorylation-dependent targeting of calcium/calmodulin-dependent protein kinase II by the NR2B subunit of the N-methyl-D-aspartate receptor. <source>J. Biol. Chem.</source>
<volume>273</volume>:<fpage>20689</fpage>–20692.
<pub-id pub-id-type="pmid">9694809</pub-id></mixed-citation></ref><ref id="bib63"><mixed-citation publication-type="journal">Strack, S., R.B. McNeill, and R.J. Colbran. <year>2000</year>. Mechanism and regulation of calcium/calmodulin-dependent protein kinase II targeting to the NR2B subunit of the N-methyl-D-aspartate receptor. <source>J. Biol. Chem.</source>
<volume>275</volume>:<fpage>23798</fpage>–23806.
<pub-id pub-id-type="pmid">10764765</pub-id></mixed-citation></ref><ref id="bib64"><mixed-citation publication-type="journal">Tavalin, S.J., R.S. Westphal, M. Colledge, L.K. Langeberg, and J.D. Scott. <year>1999</year>. The molecular architecture of neuronal kinase/phosphatase signalling complexes. <source>Biochem. Soc. Trans.</source>
<volume>27</volume>:<fpage>539</fpage>–542.
<pub-id pub-id-type="pmid">10917637</pub-id></mixed-citation></ref><ref id="bib65"><mixed-citation publication-type="journal">Vinogradova, T.M., Y.-Y. Zhou, K.Y. Bogdanov, D. Yang, M. Kuschel, H. Cheng, and R.-P. Xiao. <year>2000</year>. Sinoatrial node pacemaker activity requires Ca<sup>2+</sup>/calmodulin-dependent protein kinase II activation. <source>Circ. Res.</source>
<volume>87</volume>:<fpage>760</fpage>–767.
<pub-id pub-id-type="pmid">11055979</pub-id></mixed-citation></ref><ref id="bib66"><mixed-citation publication-type="journal">West, A.E., E.C. Griffith, and M.E. Greenberg. <year>2002</year>. Regulation of transcription factors by neuronal activity. <source>Nat. Rev. Neurosci.</source>
<volume>3</volume>:<fpage>921</fpage>–931.
<pub-id pub-id-type="pmid">12461549</pub-id></mixed-citation></ref><ref id="bib67"><mixed-citation publication-type="journal">Wolfe, J.T., H. Wang, E. Perez-Reyes, and P.Q. Barrett. <year>2002</year>. Stimulation of recombinant Ca<sub>v</sub>3.2, T-type, Ca<sup>2+</sup> channel currents by CaMKIIγ<sub>C</sub>. <source>J. Physiol.</source>
<volume>538</volume>:<fpage>343</fpage>–355.
<pub-id pub-id-type="pmid">11790804</pub-id></mixed-citation></ref><ref id="bib68"><mixed-citation publication-type="journal">Xiao, R.P., H. Cheng, W.J. Lederer, T. Suzuki, and E.G. Lakatta. <year>1994</year>. Dual regulation of Ca<sup>2+</sup>/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx. <source>Proc. Natl. Acad. Sci. USA.</source>
<volume>91</volume>:<fpage>9659</fpage>–9663.
<pub-id pub-id-type="pmid">7937825</pub-id></mixed-citation></ref><ref id="bib69"><mixed-citation publication-type="journal">Yuan, W., and D.M. Bers. <year>1994</year>. Ca-dependent facilitation of cardiac Ca current is due to Ca-calmodulin-dependent protein kinase. <source>Am. J. Physiol.</source>
<volume>267</volume>:<fpage>H982</fpage>–H993.
<pub-id pub-id-type="pmid">8092302</pub-id></mixed-citation></ref><ref id="bib70"><mixed-citation publication-type="journal">Zhang, T., E.N. Johnson, Y. Gu, M.R. Morissette, V.P. Sah, M.S. Gigena, D.D. Belke, W.H. Dillmann, T.B. Rogers, H. Schulman, et al. <year>2002</year>. The cardiac-specific nuclear δ<sub>B</sub> isoform of Ca<sup>2+</sup>/calmodulin-dependent protein kinase II induces hypertrophy and dilated cardiomyopathy associated with increased protein phosphatase 2A activity. <source>J. Biol. Chem.</source>
<volume>277</volume>:<fpage>1261</fpage>–1267.
<pub-id pub-id-type="pmid">11694533</pub-id></mixed-citation></ref><ref id="bib71"><mixed-citation publication-type="journal">Zühlke, R.D., G.S. Pitt, K. Deisseroth, R.W. Tsien, and H. Reuter. <year>1999</year>. Calmodulin supports both inactivation and facilitation of L-type calcium channels. <source>Nature.</source>
<volume>399</volume>:<fpage>159</fpage>–162.
<pub-id pub-id-type="pmid">10335846</pub-id></mixed-citation></ref><ref id="bib72"><mixed-citation publication-type="journal">Zühlke, R.D., G.S. Pitt, R.W. Tsien, and H. Reuter. <year>2000</year>. Ca<sup>2+</sup>-sensitive inactivation and facilitation of L-type Ca<sup>2+</sup> channels both depend on specific amino acid residues in a consensus calmodulin-binding motif in the α<sub>1C</sub> subunit. <source>J. Biol. Chem.</source>
<volume>275</volume>:<fpage>21121</fpage>–21129.
<pub-id pub-id-type="pmid">10779517</pub-id></mixed-citation></ref><ref id="bib73"><mixed-citation publication-type="journal">Zygmunt, A.C., and J. Maylie. <year>1990</year>. Stimulation-dependent facilitation of the high threshold calcium current in guinea-pig ventricular myocytes. <source>J. Physiol.</source>
<volume>428</volume>:<fpage>653</fpage>–671.
<pub-id pub-id-type="pmid">2172526</pub-id></mixed-citation></ref></ref-list></back></article>