Davis Jonathan P, Norman Catalina, Kobayashi Tomoyoshi, Solaro R John, Swartz Darl R, Tikunova Svetlana B
Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210, USA.
Biophys J. 2007 May 1;92(9):3195-206. doi: 10.1529/biophysj.106.095406. Epub 2007 Feb 9.
Understanding the effects of thin and thick filament proteins on the kinetics of Ca(2+) exchange with cardiac troponin C is essential to elucidating the Ca(2+)-dependent mechanisms controlling cardiac muscle contraction and relaxation. Unlike labeling of the endogenous Cys-84, labeling of cardiac troponin C at a novel engineered Cys-53 with 2-(4'-iodoacetamidoanilo)napthalene-6-sulfonic acid allowed us to accurately measure the rate of calcium dissociation from the regulatory domain of troponin C upon incorporation into the troponin complex. Neither tropomyosin nor actin alone affected the Ca(2+) binding properties of the troponin complex. However, addition of actin-tropomyosin to the troponin complex decreased the Ca(2+) sensitivity ( approximately 7.4-fold) and accelerated the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin filaments (actin-tropomyosin-troponin) increased the Ca(2+) sensitivity ( approximately 6.2-fold) and decreased the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 8.1-fold), which was completely reversed by ATP. Consistent with physiological data, replacement of cardiac troponin I with slow skeletal troponin I led to higher Ca(2+) sensitivities and slower Ca(2+) dissociation rates from troponin C in all the systems studied. Thus, both thin and thick filament proteins influence the ability of cardiac troponin C to sense and respond to Ca(2+). These results imply that both cross-bridge kinetics and Ca(2+) dissociation from troponin C work together to modulate the rate of cardiac muscle relaxation.
了解细肌丝和粗肌丝蛋白对心肌肌钙蛋白C与Ca(2+)交换动力学的影响，对于阐明控制心肌收缩和舒张的Ca(2+)依赖性机制至关重要。与内源性半胱氨酸-84的标记不同，用2-(4'-碘乙酰氨基苯胺基)萘-6-磺酸对新型工程化半胱氨酸-53处的心肌肌钙蛋白C进行标记，使我们能够准确测量肌钙蛋白C掺入肌钙蛋白复合物后从其调节结构域解离钙的速率。单独的原肌球蛋白或肌动蛋白都不会影响肌钙蛋白复合物的Ca(2+)结合特性。然而，向肌钙蛋白复合物中添加肌动蛋白-原肌球蛋白会降低Ca(2+)敏感性（约7.4倍），并加速Ca(2+)从肌钙蛋白C调节结构域的解离速率（约2.5倍）。随后向重组细肌丝（肌动蛋白-原肌球蛋白-肌钙蛋白）中添加肌球蛋白S1会增加Ca(2+)敏感性（约6.2倍），并降低Ca(2+)从肌钙蛋白C调节结构域的解离速率（约8.1倍），而ATP可使其完全逆转。与生理学数据一致，在所有研究的系统中，用慢肌骨骼肌肌钙蛋白I替代心肌肌钙蛋白I会导致更高的Ca(2+)敏感性和从肌钙蛋白C解离Ca(2+)的速率更慢。因此，细肌丝和粗肌丝蛋白都会影响心肌肌钙蛋白C感知和响应Ca(2+)的能力。这些结果表明，横桥动力学和Ca(2+)从肌钙蛋白C的解离共同作用来调节心肌舒张速率。
