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Dual mechanism of a natural CaMKII inhibitor.

ABSTRACT: Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of cellular Ca(2+) signaling. Several inhibitors are commonly used to study CaMKII function, but these inhibitors all lack specificity. CaM-KIIN is a natural, specific CaMKII inhibitor protein. CN21 (derived from CaM-KIIN amino acids 43-63) showed full specificity and potency of CaMKII inhibition. CNs completely blocked Ca(2+)-stimulated and autonomous substrate phosphorylation by CaMKII and autophosphorylation at T305. However, T286 autophosphorylation (the autophosphorylation generating autonomous activity) was only mildly affected. Two mechanisms can explain this unusual differential inhibitor effect. First, CNs inhibited activity by interacting with the CaMKII T-site (and thereby also interfered with NMDA-type glutamate receptor binding to the T-site). Because of this, the CaMKII region surrounding T286 competed with CNs for T-site interaction, whereas other substrates did not. Second, the intersubunit T286 autophosphorylation requires CaM binding both to the "kinase" and the "substrate" subunit. CNs dramatically decreased CaM dissociation, thus facilitating the ability of CaM to make T286 accessible for phosphorylation. Tat-fusion made CN21 cell penetrating, as demonstrated by a strong inhibition of filopodia motility in neurons and insulin secrection from isolated Langerhans' islets. These results reveal the inhibitory mechanism of CaM-KIIN and establish a powerful new tool for dissecting CaMKII function.

SUBMITTER: Vest RS

PROVIDER: S-EPMC2096578 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Dual mechanism of a natural CaMKII inhibitor.

Vest Rebekah S RS Davies Kurtis D KD O'Leary Heather H Port J David JD Bayer K Ulrich KU

Molecular biology of the cell 20071017 12

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of cellular Ca(2+) signaling. Several inhibitors are commonly used to study CaMKII function, but these inhibitors all lack specificity. CaM-KIIN is a natural, specific CaMKII inhibitor protein. CN21 (derived from CaM-KIIN amino acids 43-63) showed full specificity and potency of CaMKII inhibition. CNs completely blocked Ca(2+)-stimulated and autonomous substrate phosphorylation by CaMKII and autophosphorylation at T ...[more]

PMID: 17942605

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Project description:OBJECTIVE:Pathologically increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the associated Ca2+-leak from the sarcoplasmic reticulum are recognized to be important novel pharmacotherapeutic targets in heart failure and cardiac arrhythmias. However, CaMKII-inhibitory compounds for therapeutic use are still lacking. We now report on the cellular and molecular effects of a novel pyrimidine-based CaMKII inhibitor developed towards clinical use. METHODS AND RESULTS:Our findings demonstrate that AS105 is a high-affinity ATP-competitive CaMKII-inhibitor that by its mode of action is also effective against autophosphorylated CaMKII (in contrast to the commonly used allosteric CaMKII-inhibitor KN-93). In isolated atrial cardiomyocytes from human donors and ventricular myocytes from CaMKII?C-overexpressing mice with heart failure, AS105 effectively reduced diastolic SR Ca2+ leak by 38% to 65% as measured by Ca2+-sparks or tetracaine-sensitive shift in [Ca2+]i. Consistent with this, we found that AS105 suppressed arrhythmogenic spontaneous cardiomyocyte Ca2+-release (by 53%). Also, the ability of the SR to accumulate Ca2+ was enhanced by AS105, as indicated by improved post-rest potentiation of Ca2+-transient amplitudes and increased SR Ca2+-content in the murine cells. Accordingly, these cells had improved systolic Ca2+-transient amplitudes and contractility during basal stimulation. Importantly, CaMKII inhibition did not compromise systolic fractional Ca2+-release, diastolic SR Ca2+-reuptake via SERCA2a or Ca2+-extrusion via NCX. CONCLUSION:AS105 is a novel, highly potent ATP-competitive CaMKII inhibitor. In vitro, it effectively reduced SR Ca2+-leak, thus improving SR Ca2+-accumulation and reducing cellular arrhythmogenic correlates, without negatively influencing excitation-contraction coupling. These findings further validate CaMKII as a key target in cardiovascular disease, implicated by genetic, allosteric inhibitors, and pseudo-substrate inhibitors.

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Dual mechanism of a natural CaMKII inhibitor.

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Dual mechanism of a natural CaMKII inhibitor.

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