Westfall M V, Albayya F P, Metzger J M
Department of Physiology, School of Medicine, University of Michigan, Ann Arbor, Michigan 48109-0622, USA.
J Biol Chem. 1999 Aug 6;274(32):22508-16. doi: 10.1074/jbc.274.32.22508.
Troponin I is the putative molecular switch for Ca(2+)-activated contraction within the myofilament of striated muscles. To gain insight into functional troponin I domain(s) in the context of the intact myofilament, adenovirus-mediated gene transfer was used to replace endogenous cardiac troponin I within the myofilaments of adult cardiac myocytes with the slow skeletal isoform or a chimera of the slow skeletal and cardiac isoforms. Efficient expression and myofilament incorporation were observed in myocytes with each exogenous troponin I protein without detected changes in the stoichiometry of other contractile proteins and/or sarcomere architecture. Contractile function studies in single, permeabilized myocytes expressing exogenous troponin I provided support for the presence of a Ca(2+)-sensitive regulatory domain in the carboxyl terminus of troponin I and a second, newly defined Ca(2+)-sensitive domain residing in the amino terminus of troponin I. Additional experiments demonstrated that the isoform-specific, acidic pH-induced contractile dysfunction in myocytes appears to lie in the carboxyl terminus of troponin I. Functional results obtained from adult cardiac myocytes expressing the chimera or isoforms of troponin I now define multiple troponin I regulatory domains operating in the intact myofilament and provide new insight into the Ca(2+)-sensitive properties of troponin I during contraction.
肌钙蛋白I是横纹肌肌丝内Ca(2+)激活收缩的假定分子开关。为了深入了解完整肌丝背景下功能性肌钙蛋白I结构域，利用腺病毒介导的基因转移，用慢肌型或慢肌型与心肌型的嵌合体替代成年心肌细胞肌丝中的内源性心肌肌钙蛋白I。在每个外源性肌钙蛋白I蛋白的心肌细胞中均观察到有效表达和肌丝掺入，且未检测到其他收缩蛋白的化学计量和/或肌节结构发生变化。对表达外源性肌钙蛋白I的单个通透心肌细胞进行的收缩功能研究支持了肌钙蛋白I羧基末端存在Ca(2+)敏感调节结构域，以及肌钙蛋白I氨基末端存在第二个新定义的Ca(2+)敏感结构域。进一步的实验表明，心肌细胞中同工型特异性、酸性pH诱导的收缩功能障碍似乎位于肌钙蛋白I的羧基末端。从表达肌钙蛋白I嵌合体或同工型的成年心肌细胞获得的功能结果，现在定义了在完整肌丝中起作用的多个肌钙蛋白I调节结构域，并为收缩过程中肌钙蛋白I的Ca(2+)敏感特性提供了新的见解。
