Structural basis for the in situ Ca²⁺ sensitization of cardiac troponin C by positive feedback from force-generating myosin cross-bridges

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• 作者：Daniel C. Rieck ; King-Lun Li ; Yexin Ouyang ; R. John Solaro ; Wen-Ji Dong
• 关键词：Cardiac troponin ; Thin filament regulation ; Myosin cross-bridges ; FRET ; Cardiac muscle
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：2013
• 出版时间：15 September, 2013
• 年：2013
• 卷：537
• 期：2
• 页码：198-209
• 全文大小：2271 K

文摘

The in situ structural coupling between the cardiac troponin (cTn) Ca²⁺-sensitive regulatory switch (CRS) and strong myosin cross-bridges was investigated using F?rster resonance energy transfer (FRET). The double cysteine mutant cTnC(T13C/N51C) was fluorescently labeled with the FRET pair 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid (IAEDENS) and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and then incorporated into detergent skinned left ventricular papillary fiber bundles. Ca²⁺ titrations of cTnC(T13C/N51C)_AEDENS/DDPM-reconstituted fibers showed that the Ca²⁺-dependence of the opening of the N-domain of cTnC (N-cTnC) statistically matched the force?Ca²⁺ relationship. N-cTnC opening still occurred steeply during Ca²⁺ titrations in the presence of 1 mM vanadate, but the maximal extent of ensemble-averaged N-cTnC opening and the Ca²⁺-sensitivity of the CRS were significantly reduced. At nanomolar, resting Ca²⁺ levels, treatment with ADP¡¤Mg in the absence of ATP caused a partial opening of N-cTnC. During subsequent Ca²⁺ titrations in the presence of ADP¡¤Mg and absence of ATP, further N-cTnC opening was stimulated as the CRS responded to Ca²⁺ with increased Ca²⁺-sensitivity and reduced steepness. These findings supported our hypothesis here that strong cross-bridge interactions with the cardiac thin filament exert a Ca²⁺-sensitizing effect on the CRS by stabilizing the interaction between the exposed hydrophobic patch of N-cTnC and the switch region of cTnI.