Mathur Mohit C, Kobayashi Tomoyoshi, Chalovich Joseph M
Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina, USA.
Biophys J. 2008 Jan 15;94(2):542-9. doi: 10.1529/biophysj.107.113944. Epub 2007 Sep 14.
Alterations in the troponin complex can lead to increases or decreases in contractile activity. Most mutations of troponin that cause hypertrophic cardiomyopathy increase the activity of cardiac muscle fibers. In at least some cases these mutants stabilize the active state of regulated actin. In contrast, phosphorylation of troponin I at residues 43, 45, and 144 inhibits muscle contractility. To determine if alterations of troponin I that reduce activity do stabilize the inactive state of actin, we introduced negative charges at residues 43, 45, and 144 of troponin I to mimic a constitutively phosphorylated state. At saturating calcium, all mutants decreased ATPase rates relative to wild-type actin-tropomyosin-troponin. Reduced activation of ATPase activity was seen with a single mutation at S45E and was not further altered by mutating the other two sites. In the presence of low concentrations of NEM-S1, wild-type troponin was more active than the mutants. At high NEM-S1, the rates of wild-type and mutants approached the same limiting value. Changes in Ca(2+) affinity also support the idea that the equilibrium between states of actin-tropomyosin-troponin was shifted to the inactive state by mutations that mimic troponin I phosphorylation.
肌钙蛋白复合物的改变可导致收缩活性的增加或降低。大多数导致肥厚型心肌病的肌钙蛋白突变会增加心肌纤维的活性。至少在某些情况下，这些突变体可稳定调节型肌动蛋白的活性状态。相反，肌钙蛋白I第43、45和144位残基的磷酸化会抑制肌肉收缩力。为了确定降低活性的肌钙蛋白I改变是否确实稳定了肌动蛋白的非活性状态，我们在肌钙蛋白I的第43、45和144位残基引入负电荷以模拟持续磷酸化状态。在钙饱和时，相对于野生型肌动蛋白-原肌球蛋白-肌钙蛋白，所有突变体均降低了ATP酶活性。在S45E处单一位点突变时可观察到ATP酶活性激活降低，而突变其他两个位点并未使其进一步改变。在低浓度NEM-S1存在时，野生型肌钙蛋白比突变体更具活性。在高浓度NEM-S1时，野生型和突变体的活性速率接近相同的极限值。Ca(2+)亲和力的变化也支持这样一种观点，即模拟肌钙蛋白I磷酸化状态的突变会使肌动蛋白-原肌球蛋白-肌钙蛋白状态之间的平衡向非活性状态转变。
