The CaMKII inhibitor KN93-calmodulin interaction and implications for calmodulin tuning of NaV1.5 and RyR2 function

Christopher N. Johnson; R. Pattanayek; Franck Potet; Robyn T. Rebbeck; Daniel J. Blackwell; R. Nikolaienko; V. Sequeira; Remy Le Meur; Przemysław B. Radwański; Jonathan P. Davis; Aleksey V. Zima; Razvan L. Cornea; Steven M. Damo; Sandor Györke; Alfred L. George; Björn C. Knollmann

doi:10.1016/j.ceca.2019.102063

The CaMKII inhibitor KN93-calmodulin interaction and implications for calmodulin tuning of Na_V1.5 and RyR2 function

Christopher N. Johnson, R. Pattanayek, Franck Potet, Robyn T. Rebbeck, Daniel J. Blackwell, R. Nikolaienko, V. Sequeira, Remy Le Meur, Przemysław B. Radwański, Jonathan P. Davis, Aleksey V. Zima, Razvan L. Cornea, Steven M. Damo, Sandor Györke, Alfred L. George, Björn C. Knollmann

Chemical and Structural Biology (TMED)

Research output: Contribution to journal › Article › peer-review

26 Scopus citations

Abstract

Here we report the structure of the widely utilized calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitor KN93 bound to the Ca²⁺-sensing protein CaM. KN93 is widely believed to inhibit CaMKII by binding to the kinase. The CaM-KN93 interaction is significant as it can interfere with the interaction between CaM and it's physiological targets, thereby raising the possibility of ascribing modified protein function to CaMKII phosphorylation while concealing a CaM–protein interaction. NMR spectroscopy, stopped-flow kinetic measurements, and x-ray crystallography were used to characterize the structure and biophysical properties of the CaM-KN93 interaction. We then investigated the functional properties of the cardiac Na⁺ channel (Na_V1.5) and ryanodine receptor (RyR2). We find that KN93 disrupts a high affinity CaM-Na_V1.5 interaction and alters channel function independent of CaMKII. Moreover, KN93 increases RyR2 Ca²⁺ release in cardiomyocytes independent of CaMKII. Therefore, when interpreting KN93 data, targets other than CaMKII need to be considered.

Original language	English (US)
Article number	102063
Journal	Cell Calcium
Volume	82
DOIs	https://doi.org/10.1016/j.ceca.2019.102063
State	Published - Sep 2019

Bibliographical note

Funding Information:
We thank Dr. Christopher George (University of Cardiff, UK) for providing cDNA of the human ryanodine receptor type 2 (RyR2) fused with amino-terminal GFP tag (GFP-RyR2). We thank Walter Chazin for provided resources for recombinant protein production and NMR time (NIH R35 GM118089). This work was also supported in part by: the National Science Foundation (NSF) grant HRD-1547757 (S.M.D.), grants for NMR instrumentation from the NSF (0922862), NIH (S10 RR025677), and Vanderbilt University matching funds. Imaging was supported by Vanderbilt University Cell Imaging Shared Resources. NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126. NIH HL138579 (S.G. J.P.D.) and HL127299 (P.B.R.) American Heart Association Postdoctoral Fellowship (16POST31010019) (R.T.R.). NIH grants R01HL092097 and R01HL138539 (R.L.C.) Foundation Leducq (A.L.G. F.P.), NIH T32 NS007491 (D.J.B.), R01 HL130231 (to A.V.Z.) Argonne National Laboratory contract DE-AC02-06CH11357. Use of LS-CAT Sector 21 supported by the Michigan Economic Development Corporation (Grant 085P100817).

Funding Information:
We thank Dr. Christopher George (University of Cardiff, UK) for providing cDNA of the human ryanodine receptor type 2 (RyR2) fused with amino-terminal GFP tag (GFP-RyR2). We thank Walter Chazin for provided resources for recombinant protein production and NMR time (NIH R35 GM118089). This work was also supported in part by: the National Science Foundation (NSF) grant HRD-1547757 (S.M.D.), grants for NMR instrumentation from the NSF ( 0922862 ), NIH ( S10 RR025677 ), and Vanderbilt University matching funds . Imaging was supported by Vanderbilt University Cell Imaging Shared Resources. NIH grants CA68485 , DK20593 , DK58404 , DK59637 and EY08126 . NIH HL138579 (S.G. J.P.D.) and HL127299 (P.B.R.) American Heart Association Postdoctoral Fellowship ( 16POST31010019 ) (R.T.R.). NIH grants R01HL092097 and R01HL138539 (R.L.C.) Foundation Leducq (A.L.G., F.P.), NIH T32 NS007491 (D.J.B.), R01 HL130231 (to A.V.Z.) Argonne National Laboratory contract DE-AC02-06CH11357 . Use of LS-CAT Sector 21 supported by the Michigan Economic Development Corporation (Grant 085P100817 ).

Publisher Copyright:
© 2019 Elsevier Ltd

Keywords

CaMKII inhibition
Calcium calmodulin dependent kinase type II (CaMKII)
Calcium signaling
KN92
KN93
autocamtide-2-inhibitor peptide

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10.1016/j.ceca.2019.102063

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Johnson, C. N., Pattanayek, R., Potet, F., Rebbeck, R. T., Blackwell, D. J., Nikolaienko, R., Sequeira, V., Le Meur, R., Radwański, P. B., Davis, J. P., Zima, A. V., Cornea, R. L., Damo, S. M., Györke, S., George, A. L., & Knollmann, B. C. (2019). The CaMKII inhibitor KN93-calmodulin interaction and implications for calmodulin tuning of Na_V1.5 and RyR2 function. Cell Calcium, 82, Article 102063. https://doi.org/10.1016/j.ceca.2019.102063

Johnson, CN, Pattanayek, R, Potet, F, Rebbeck, RT, Blackwell, DJ, Nikolaienko, R, Sequeira, V, Le Meur, R, Radwański, PB, Davis, JP, Zima, AV, Cornea, RL, Damo, SM, Györke, S, George, AL & Knollmann, BC 2019, 'The CaMKII inhibitor KN93-calmodulin interaction and implications for calmodulin tuning of Na_V1.5 and RyR2 function', Cell Calcium, vol. 82, 102063. https://doi.org/10.1016/j.ceca.2019.102063

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title = "The CaMKII inhibitor KN93-calmodulin interaction and implications for calmodulin tuning of NaV1.5 and RyR2 function",

abstract = "Here we report the structure of the widely utilized calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitor KN93 bound to the Ca2+-sensing protein CaM. KN93 is widely believed to inhibit CaMKII by binding to the kinase. The CaM-KN93 interaction is significant as it can interfere with the interaction between CaM and it's physiological targets, thereby raising the possibility of ascribing modified protein function to CaMKII phosphorylation while concealing a CaM–protein interaction. NMR spectroscopy, stopped-flow kinetic measurements, and x-ray crystallography were used to characterize the structure and biophysical properties of the CaM-KN93 interaction. We then investigated the functional properties of the cardiac Na+ channel (NaV1.5) and ryanodine receptor (RyR2). We find that KN93 disrupts a high affinity CaM-NaV1.5 interaction and alters channel function independent of CaMKII. Moreover, KN93 increases RyR2 Ca2+ release in cardiomyocytes independent of CaMKII. Therefore, when interpreting KN93 data, targets other than CaMKII need to be considered.",

keywords = "CaMKII inhibition, Calcium calmodulin dependent kinase type II (CaMKII), Calcium signaling, KN92, KN93, autocamtide-2-inhibitor peptide",

author = "Johnson, {Christopher N.} and R. Pattanayek and Franck Potet and Rebbeck, {Robyn T.} and Blackwell, {Daniel J.} and R. Nikolaienko and V. Sequeira and {Le Meur}, Remy and Radwa{\'n}ski, {Przemys{\l}aw B.} and Davis, {Jonathan P.} and Zima, {Aleksey V.} and Cornea, {Razvan L.} and Damo, {Steven M.} and Sandor Gy{\"o}rke and George, {Alfred L.} and Knollmann, {Bj{\"o}rn C.}",

note = "Funding Information: We thank Dr. Christopher George (University of Cardiff, UK) for providing cDNA of the human ryanodine receptor type 2 (RyR2) fused with amino-terminal GFP tag (GFP-RyR2). We thank Walter Chazin for provided resources for recombinant protein production and NMR time (NIH R35 GM118089). This work was also supported in part by: the National Science Foundation (NSF) grant HRD-1547757 (S.M.D.), grants for NMR instrumentation from the NSF (0922862), NIH (S10 RR025677), and Vanderbilt University matching funds. Imaging was supported by Vanderbilt University Cell Imaging Shared Resources. NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126. NIH HL138579 (S.G. J.P.D.) and HL127299 (P.B.R.) American Heart Association Postdoctoral Fellowship (16POST31010019) (R.T.R.). NIH grants R01HL092097 and R01HL138539 (R.L.C.) Foundation Leducq (A.L.G. F.P.), NIH T32 NS007491 (D.J.B.), R01 HL130231 (to A.V.Z.) Argonne National Laboratory contract DE-AC02-06CH11357. Use of LS-CAT Sector 21 supported by the Michigan Economic Development Corporation (Grant 085P100817). Funding Information: We thank Dr. Christopher George (University of Cardiff, UK) for providing cDNA of the human ryanodine receptor type 2 (RyR2) fused with amino-terminal GFP tag (GFP-RyR2). We thank Walter Chazin for provided resources for recombinant protein production and NMR time (NIH R35 GM118089). This work was also supported in part by: the National Science Foundation (NSF) grant HRD-1547757 (S.M.D.), grants for NMR instrumentation from the NSF ( 0922862 ), NIH ( S10 RR025677 ), and Vanderbilt University matching funds . Imaging was supported by Vanderbilt University Cell Imaging Shared Resources. NIH grants CA68485 , DK20593 , DK58404 , DK59637 and EY08126 . NIH HL138579 (S.G. J.P.D.) and HL127299 (P.B.R.) American Heart Association Postdoctoral Fellowship ( 16POST31010019 ) (R.T.R.). NIH grants R01HL092097 and R01HL138539 (R.L.C.) Foundation Leducq (A.L.G., F.P.), NIH T32 NS007491 (D.J.B.), R01 HL130231 (to A.V.Z.) Argonne National Laboratory contract DE-AC02-06CH11357 . Use of LS-CAT Sector 21 supported by the Michigan Economic Development Corporation (Grant 085P100817 ). Publisher Copyright: {\textcopyright} 2019 Elsevier Ltd",

year = "2019",

month = sep,

doi = "10.1016/j.ceca.2019.102063",

language = "English (US)",

volume = "82",

journal = "Cell Calcium",

issn = "0143-4160",

publisher = "Churchill Livingstone",

}

TY - JOUR

T1 - The CaMKII inhibitor KN93-calmodulin interaction and implications for calmodulin tuning of NaV1.5 and RyR2 function

AU - Johnson, Christopher N.

AU - Pattanayek, R.

AU - Potet, Franck

AU - Rebbeck, Robyn T.

AU - Blackwell, Daniel J.

AU - Nikolaienko, R.

AU - Sequeira, V.

AU - Le Meur, Remy

AU - Radwański, Przemysław B.

AU - Davis, Jonathan P.

AU - Zima, Aleksey V.

AU - Cornea, Razvan L.

AU - Damo, Steven M.

AU - Györke, Sandor

AU - George, Alfred L.

AU - Knollmann, Björn C.

N1 - Funding Information: We thank Dr. Christopher George (University of Cardiff, UK) for providing cDNA of the human ryanodine receptor type 2 (RyR2) fused with amino-terminal GFP tag (GFP-RyR2). We thank Walter Chazin for provided resources for recombinant protein production and NMR time (NIH R35 GM118089). This work was also supported in part by: the National Science Foundation (NSF) grant HRD-1547757 (S.M.D.), grants for NMR instrumentation from the NSF (0922862), NIH (S10 RR025677), and Vanderbilt University matching funds. Imaging was supported by Vanderbilt University Cell Imaging Shared Resources. NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126. NIH HL138579 (S.G. J.P.D.) and HL127299 (P.B.R.) American Heart Association Postdoctoral Fellowship (16POST31010019) (R.T.R.). NIH grants R01HL092097 and R01HL138539 (R.L.C.) Foundation Leducq (A.L.G. F.P.), NIH T32 NS007491 (D.J.B.), R01 HL130231 (to A.V.Z.) Argonne National Laboratory contract DE-AC02-06CH11357. Use of LS-CAT Sector 21 supported by the Michigan Economic Development Corporation (Grant 085P100817). Funding Information: We thank Dr. Christopher George (University of Cardiff, UK) for providing cDNA of the human ryanodine receptor type 2 (RyR2) fused with amino-terminal GFP tag (GFP-RyR2). We thank Walter Chazin for provided resources for recombinant protein production and NMR time (NIH R35 GM118089). This work was also supported in part by: the National Science Foundation (NSF) grant HRD-1547757 (S.M.D.), grants for NMR instrumentation from the NSF ( 0922862 ), NIH ( S10 RR025677 ), and Vanderbilt University matching funds . Imaging was supported by Vanderbilt University Cell Imaging Shared Resources. NIH grants CA68485 , DK20593 , DK58404 , DK59637 and EY08126 . NIH HL138579 (S.G. J.P.D.) and HL127299 (P.B.R.) American Heart Association Postdoctoral Fellowship ( 16POST31010019 ) (R.T.R.). NIH grants R01HL092097 and R01HL138539 (R.L.C.) Foundation Leducq (A.L.G., F.P.), NIH T32 NS007491 (D.J.B.), R01 HL130231 (to A.V.Z.) Argonne National Laboratory contract DE-AC02-06CH11357 . Use of LS-CAT Sector 21 supported by the Michigan Economic Development Corporation (Grant 085P100817 ). Publisher Copyright: © 2019 Elsevier Ltd

PY - 2019/9

Y1 - 2019/9

N2 - Here we report the structure of the widely utilized calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitor KN93 bound to the Ca2+-sensing protein CaM. KN93 is widely believed to inhibit CaMKII by binding to the kinase. The CaM-KN93 interaction is significant as it can interfere with the interaction between CaM and it's physiological targets, thereby raising the possibility of ascribing modified protein function to CaMKII phosphorylation while concealing a CaM–protein interaction. NMR spectroscopy, stopped-flow kinetic measurements, and x-ray crystallography were used to characterize the structure and biophysical properties of the CaM-KN93 interaction. We then investigated the functional properties of the cardiac Na+ channel (NaV1.5) and ryanodine receptor (RyR2). We find that KN93 disrupts a high affinity CaM-NaV1.5 interaction and alters channel function independent of CaMKII. Moreover, KN93 increases RyR2 Ca2+ release in cardiomyocytes independent of CaMKII. Therefore, when interpreting KN93 data, targets other than CaMKII need to be considered.

AB - Here we report the structure of the widely utilized calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitor KN93 bound to the Ca2+-sensing protein CaM. KN93 is widely believed to inhibit CaMKII by binding to the kinase. The CaM-KN93 interaction is significant as it can interfere with the interaction between CaM and it's physiological targets, thereby raising the possibility of ascribing modified protein function to CaMKII phosphorylation while concealing a CaM–protein interaction. NMR spectroscopy, stopped-flow kinetic measurements, and x-ray crystallography were used to characterize the structure and biophysical properties of the CaM-KN93 interaction. We then investigated the functional properties of the cardiac Na+ channel (NaV1.5) and ryanodine receptor (RyR2). We find that KN93 disrupts a high affinity CaM-NaV1.5 interaction and alters channel function independent of CaMKII. Moreover, KN93 increases RyR2 Ca2+ release in cardiomyocytes independent of CaMKII. Therefore, when interpreting KN93 data, targets other than CaMKII need to be considered.

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KW - Calcium calmodulin dependent kinase type II (CaMKII)

KW - Calcium signaling

KW - KN92

KW - KN93

KW - autocamtide-2-inhibitor peptide

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