Hudmon A , Schulman H , Kim J , Maltez JM , Tsien RW , Pitt GS .

K08 HL003743-05 NHLBI NIH HHS , K08 HL003743-06 NHLBI NIH HHS , R01 GM058234-05 NIGMS NIH HHS , R01 GM058234-06 NIGMS NIH HHS , R01 GM058234-07 NIGMS NIH HHS , R01 HL071165-01 NHLBI NIH HHS , R01 HL071165-02 NHLBI NIH HHS , R01 HL071165-03 NHLBI NIH HHS , R01 HL071165-04 NHLBI NIH HHS , R01 NS024067 NINDS NIH HHS , R01 GM058234 NIGMS NIH HHS , K08 HL003743 NHLBI NIH HHS , R01 HL071165 NHLBI NIH HHS

	Figure 1. Phosphorylation of the α1C subunit by CaMKII. (A) Autoradiogram showing phosphorylation of α1C by CaMKII. Lysates from HEK cells transfected with α1C and β2b (lanes 2–4) or nontransfected cells (lane 1) were immunoprecipitated with an anti-α1C antibody (lanes 1, 3, and 4) or control IgG (lane 2) and then incubated with purified α-CaMKII in the presence of Ca2+/CaM and Mg2+/ATP32 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 α1C 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-α1C immunoblot of the samples used in the kinase reaction is shown below the autoradiogram. (B) Schematic of α1C. 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 Ca2+/CaM and Mg2+/ATP32 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.
	Figure 2. CaMKII coimmunoprecipitates and colocalizes with α1C. (A) Biotinylated calmodulin overlay of rat cardiac sarcolemmal membranes after immunoprecipitation with an anti-α1C antibody. Purified α-CaMKII was run as a control to demonstrate effectiveness of CaM overlay. An anti-α1C 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-α1C antibody (lane 5) from lysates of HEK 293 cells transiently transfected with GFP-CaMKII and α1C.
	Figure 3. Activity-dependent interaction of CaMKII with the cytoplasmic determinants of α1C. Immunoblots using an mAb (CBα2) for CaMKII after a GST pull-down assay with 20 nM of native (top), Ca2+/CaM-activated (middle), or Ca2+/CaM/autophosphorylated α-CaMKII (bottom). GST fusion proteins contained various cytoplasmic regions of α1C just as in Fig. 1 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.
	Figure 4. Localization of the CaMKII binding site on the COOH terminus of α1C. (A) Diagram of α1C 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 α1C 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 α1C aa 1581–1690 (wild type [WT]), a 1644TVGKFY1649 → 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 (n = 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.
	Figure 5. CaMKII interaction with the COOH terminus of α1C is essential for CDF. (A) IBa and scaled ICa traces during a train of 40 test pulses of Vh from –90 mV to +20 mV at 3.3 Hz recorded from oocytes expressing α1C I1654A (I/A) or α1C I1654A/1644TVGKFY1649 → EEDAAA (I/A-Mut6). Bars, 500 nA and 25 ms. (B) Peak IBa and ICa during trains of 40 repetitive test pulses at 3.3 Hz, normalized to the current amplitude at the beginning of each train (n = 4–5). Values indicate means ± SEM. (C) Changes in peak IBa and ICa conducted by α1C I1654A (I/A) or α1C I1654A/1644TVGKFY1649 → EEDAAA (I/A-Mut6) at indicated stimulation frequencies (n = 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 α1C (Mut6) by CaMKII, performed as in Fig. 1 A. An anti-α1C immunoblot of the samples used in the kinase reaction confirmed similar expression levels of the WT and mutant α1C subunits. An anti-CaMKII immunoblot with CBα2 confirmed the identity of the retained 50-kD 32P-labeled protein as α-CaMKII.
	Figure 6. The binding site for the COOH terminus of α1C on CaMKII is localized near the catalytic domain. (A) Biotinylated CaM overlay of GST pull downs, using a fusion protein from the COOH terminus of α1C (aa 1509–1905) on lysates of HEK 293 cells transiently transfected with the CaMKII isoforms (α, β, δA, δC, and γB; 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 α1c (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 α1C with the autoregulatory domain from α-CaMKII.
	Figure 7. CaMKII interaction with the COOH terminus of α1C is not reversed by dephosphorylation or CaM dissociation, and tethered CaMKII requires autophosphorylation or Ca2+/CaM for activity. (A and B) Immunoblots with CBα2 or a phosphospecific CaMKII mAb after GST pull-down assays, using α1C 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 (n = 4). (D) Activity measurements, using peptide AC-2 as a substrate, of CaMKII recovered in GST pull-down assays, using α1C aa 1509–1905. Ca2+/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 (n = 4) Values indicate means ± SD.
	Figure 8. Proposed mechanism of CaMKII binding to α1C to form a local and dedicated Ca2+ spike integrator for CDF. A catalytic core and autoregulatory domain for a prototypical CaMKII inactive subunit is shown on the bottom left (inactive is indicated by green). Ca2+/CaM activation and Thr286 autophosphorylation displace the CaMKII autoregulatory domain within the catalytic lobe to activate the subunit (yellow) and to expose an α1C tethering site. The CaMKII holoenzyme remains bound to the α1C COOH terminus even after removal of the Ca2+/CaM stimulus, and CaMKII dephosphorylation produces an inactive subunit. CaMKII may remain tethered to other cytoplasmic domains of α1C as well. High depolarization frequencies would produce a threshold level of activated/autophosphorylated CaMKII subunits that increase the Po of the channel via phosphorylation of the NH2 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 Po for α1C (top right).

Anderson, Multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca(2+)-induced enhancement of the L-type Ca2+ current in rabbit ventricular myocytes. 1994, Pubmed

Anderson, Multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca(2+)-induced enhancement of the L-type Ca2+ current in rabbit ventricular myocytes. 1994, Pubmed
Bayer, Alternative splicing modulates the frequency-dependent response of CaMKII to Ca(2+) oscillations. 2002, Pubmed
Bayer, Interaction with the NMDA receptor locks CaMKII in an active conformation. 2001, Pubmed
Bence-Hanulec, Potentiation of neuronal L calcium channels by IGF-1 requires phosphorylation of the alpha1 subunit on a specific tyrosine residue. 2000, Pubmed
Bradley, An evaluation of specificity in activity-dependent gene expression in neurons. 2002, Pubmed
Bradshaw, Chemical quenched flow kinetic studies indicate an intraholoenzyme autophosphorylation mechanism for Ca2+/calmodulin-dependent protein kinase II. 2002, Pubmed
Bradshaw, An ultrasensitive Ca2+/calmodulin-dependent protein kinase II-protein phosphatase 1 switch facilitates specificity in postsynaptic calcium signaling. 2003, Pubmed
Braun, A non-selective cation current activated via the multifunctional Ca(2+)-calmodulin-dependent protein kinase in human epithelial cells. 1995, Pubmed
Bünemann, Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the beta(2) subunit. 1999, Pubmed
Cuttle, Facilitation of the presynaptic calcium current at an auditory synapse in rat brainstem. 1998, Pubmed
Deisseroth, Signaling from synapse to nucleus: the logic behind the mechanisms. 2003, Pubmed
De Jongh, Specific phosphorylation of a site in the full-length form of the alpha 1 subunit of the cardiac L-type calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. 1996, Pubmed
De Koninck, Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations. 1998, Pubmed
DeMaria, Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels. 2001, Pubmed
DeSantiago, Frequency-dependent acceleration of relaxation in the heart depends on CaMKII, but not phospholamban. 2002, Pubmed
Dolmetsch, Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. 2001, Pubmed
Dorn, Intracellular transport mechanisms of signal transducers. 2002, Pubmed
Dzhura, Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels. 2000, Pubmed
Edman, Identification and characterization of delta B-CaM kinase and delta C-CaM kinase from rat heart, two new multifunctional Ca2+/calmodulin-dependent protein kinase isoforms. 1994, Pubmed
Erickson, FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation. 2003, Pubmed
Eshete, Spike frequency decoding and autonomous activation of Ca2+-calmodulin-dependent protein kinase II in dorsal root ganglion neurons. 2001, Pubmed
Feldman, Depression of systolic and diastolic myocardial reserve during atrial pacing tachycardia in patients with dilated cardiomyopathy. 1988, Pubmed
Gao, C-terminal fragments of the alpha 1C (CaV1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated alpha 1C subunits. 2001, Pubmed
Gao, cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits. 1997, Pubmed
Glenney, Detection of calmodulin-binding polypeptides separated in SDS-polyacrylamide gels by a sensitive [125I]calmodulin gel overlay assay. 1983, Pubmed
Hanson, Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation. 1989, Pubmed
Harootunian, Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion. 1993, Pubmed
Hasenfuss, Influence of the force-frequency relationship on haemodynamics and left ventricular function in patients with non-failing hearts and in patients with dilated cardiomyopathy. 1994, Pubmed
Hell, Phosphorylation of presynaptic and postsynaptic calcium channels by cAMP-dependent protein kinase in hippocampal neurons. 1995, Pubmed
Hryshko, Ca current facilitation during postrest recovery depends on Ca entry. 1990, Pubmed
Hudmon, Neuronal CA2+/calmodulin-dependent protein kinase II: the role of structure and autoregulation in cellular function. 2002, Pubmed
Hudmon, Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II. 2002, Pubmed
Hulme, A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2+ channel and modulates its function. 2002, Pubmed
Ikeda, Studies on the generation of Ca2+/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme. 1991, Pubmed
Kim, Identification of the components controlling inactivation of voltage-gated Ca2+ channels. 2004, Pubmed , Xenbase
KOCH-WESER, THE INFLUENCE OF THE INTERVAL BETWEEN BEATS ON MYOCARDIAL CONTRACTILITY. 1963, Pubmed
Kolodziej, Three-dimensional reconstructions of calcium/calmodulin-dependent (CaM) kinase IIalpha and truncated CaM kinase IIalpha reveal a unique organization for its structural core and functional domains. 2000, Pubmed
Lai, Ca2+/calmodulin-dependent protein kinase II: identification of autophosphorylation sites responsible for generation of Ca2+/calmodulin-independence. 1987, Pubmed
Lee, Ca2+/calmodulin binds to and modulates P/Q-type calcium channels. 1999, Pubmed
Lee, Potentiation of the calcium-channel currents of internally perfused mammalian heart cells by repetitive depolarization. 1987, Pubmed
Leonard, Regulation of calcium/calmodulin-dependent protein kinase II docking to N-methyl-D-aspartate receptors by calcium/calmodulin and alpha-actinin. 2002, Pubmed
Leonard, Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-D-aspartate receptor. 1999, Pubmed
Lisman, The molecular basis of CaMKII function in synaptic and behavioural memory. 2002, Pubmed
Lou, Distinct autophosphorylation sites sequentially produce autonomy and inhibition of the multifunctional Ca2+/calmodulin-dependent protein kinase. 1989, Pubmed
Maier, Calcium, calmodulin, and calcium-calmodulin kinase II: heartbeat to heartbeat and beyond. 2002, Pubmed
Marban, Enhancement of calcium current during digitalis inotropy in mammalian heart: positive feed-back regulation by intracellular calcium? 1982, Pubmed
McCarron, Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II. 1992, Pubmed
McHugh, Inhibition of cardiac L-type calcium channels by protein kinase C phosphorylation of two sites in the N-terminal domain. 2000, Pubmed
Meyer, Calmodulin trapping by calcium-calmodulin-dependent protein kinase. 1992, Pubmed
Miller, Sequences of autophosphorylation sites in neuronal type II CaM kinase that control Ca2(+)-independent activity. 1988, Pubmed
Mulieri, Altered myocardial force-frequency relation in human heart failure. 1992, Pubmed
Noble, The calcium and frequency dependence of the slow inward current 'staircase' in frog atrium. 1981, Pubmed
Pate, Determinants for calmodulin binding on voltage-dependent Ca2+ channels. 2000, Pubmed
Peterson, Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L-type calcium channels. 1999, Pubmed
Pitt, Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels. 2001, Pubmed , Xenbase
Romanin, Ca(2+) sensors of L-type Ca(2+) channel. 2000, Pubmed
Ross, Adrenergic control of the force-frequency relation. 1995, Pubmed
Rotman, Sites of selective cAMP-dependent phosphorylation of the L-type calcium channel alpha 1 subunit from intact rabbit skeletal muscle myotubes. 1995, Pubmed
Schechtman, Adaptor proteins in protein kinase C-mediated signal transduction. 2001, Pubmed
Schouten, Regulation of Ca2+ current in frog ventricular myocytes by the holding potential, c-AMP and frequency. 1989, Pubmed
Singla, Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II. 2001, Pubmed
Strack, Autophosphorylation-dependent targeting of calcium/ calmodulin-dependent protein kinase II by the NR2B subunit of the N-methyl- D-aspartate receptor. 1998, Pubmed
Strack, Mechanism and regulation of calcium/calmodulin-dependent protein kinase II targeting to the NR2B subunit of the N-methyl-D-aspartate receptor. 2000, Pubmed
Tavalin, The molecular architecture of neuronal kinase/phosphatase signalling complexes. 1999, Pubmed
Vinogradova, Sinoatrial node pacemaker activity requires Ca(2+)/calmodulin-dependent protein kinase II activation. 2000, Pubmed
West, Regulation of transcription factors by neuronal activity. 2002, Pubmed
Wolfe, Stimulation of recombinant Ca(v)3.2, T-type, Ca(2+) channel currents by CaMKIIgamma(C). 2002, Pubmed
Xiao, Dual regulation of Ca2+/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx. 1994, Pubmed
Yuan, Ca-dependent facilitation of cardiac Ca current is due to Ca-calmodulin-dependent protein kinase. 1994, Pubmed
Zhang, The cardiac-specific nuclear delta(B) isoform of Ca2+/calmodulin-dependent protein kinase II induces hypertrophy and dilated cardiomyopathy associated with increased protein phosphatase 2A activity. 2002, Pubmed
Zühlke, Calmodulin supports both inactivation and facilitation of L-type calcium channels. 1999, Pubmed , Xenbase
Zühlke, Ca2+-sensitive inactivation and facilitation of L-type Ca2+ channels both depend on specific amino acid residues in a consensus calmodulin-binding motif in the(alpha)1C subunit. 2000, Pubmed
Zygmunt, Stimulation-dependent facilitation of the high threshold calcium current in guinea-pig ventricular myocytes. 1990, Pubmed