Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex

William Schlecht; King-Lun Li; Dehong Hu; Wenji Dong

doi:10.1111/cbdd.12651

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Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex

Journal article

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Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex

William Schlecht, King-Lun Li, Dehong Hu and Wenji Dong

Chemical biology & drug design, Vol.87(2), pp.171-181

02/2016

DOI: https://doi.org/10.1111/cbdd.12651

Handle:

https://hdl.handle.net/2376/106624

PMCID: PMC4718833

PMID: 26375298

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Abstract

Troponin - metabolism

Phosphorylation

Mutagenesis, Site-Directed

Calcium - metabolism

Troponin - genetics

Cardiotonic Agents - metabolism

Troponin - chemistry

Rats

Spectrometry, Fluorescence

Cardiotonic Agents - chemistry

Hydrazones - metabolism

Hydrazones - chemistry

Pyridazines - chemistry

Animals

Myocardium - metabolism

Bepridil - chemistry

Protein Binding

Pyridazines - metabolism

Kinetics

Bepridil - metabolism

Calcium sensitizers enhance the transduction of the Ca(2+) signal into force within the heart and have found use in treating heart failure. However the mechanisms of action for most Ca(2+) sensitizers remain unclear. To address this issue an efficient fluorescence based approach to Ca(2+) sensitizer screening was developed which monitors cardiac troponin C's (cTnC's) hydrophobic cleft. This approach was tested on four common Ca(2+) -sensitizers, EMD 57033, levosimendan, bepridil and pimobendan with the aim of elucidating the mechanisms of action for each as well as proving the efficacy of the new screening method. Ca(2+) -titration experiments were employed to determine the effect on Ca(2+) sensitivity and cooperativity of cTnC opening, while stopped flow experiments were used to investigate the impact on cTnC relaxation kinetics. Bepridil was shown to increase the sensitivity of cTnC for Ca(2+) under all reconstitution conditions, sensitization by the other drugs was context dependent. Levosimendan and pimobendan reduced the rate of cTnC closing consistent with a stabilization of cTnC's open conformation while bepridil increased the rate of relaxation. Experiments were also run on samples containing cTnT(T204E), a known Ca(2+) -desensitizing phosphorylation mimic. Levosimendan, bepridil, and pimobendan were found to elevate the Ca(2+) -sensitivity of cTnT(T204E) containing samples in this context.

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Title: Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex
Creators: William Schlecht - The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, PO Box 646515, Washington State University, Pullman, WA 99164-6515, USA
King-Lun Li - The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, PO Box 646515, Washington State University, Pullman, WA 99164-6515, USA
Dehong Hu - The Environmental and Molecular Science Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Boulevard Richland, WA 99354, USA
Wenji Dong - The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, PO Box 646515, Washington State University, Pullman, WA 99164-6515, USA
Publication Details: Chemical biology & drug design, Vol.87(2), pp.171-181
Academic Unit: Chemical Engineering and Bioengineering, School of
Publisher: England
Grant note: HL80186 / NHLBI NIH HHS T32 GM008336 / NIGMS NIH HHS 1R21HL109693 / NHLBI NIH HHS R21 HL109693 / NHLBI NIH HHS R01 HL080186 / NHLBI NIH HHS T32-GM008336 / NIGMS NIH HHS
Identifiers: 99900547077101842
Language: English
Resource Type: Journal article