Negative charges at protein kinase C sites of troponin I stabilize the inactive state of actin.
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.
Mathur
Mohit C
MC
Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, North Carolina, USA.
Kobayashi
Tomoyoshi
T
Chalovich
Joseph M
JM
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000378:metabolism; D006801:Humans; D051379:Mice; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009218:Myosins / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D013816:Thermodynamics; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000378:metabolism*
D000199:Actins; D050505:Mutant Proteins; D019209:Troponin C; D019210:Troponin I; D000244:Adenosine Diphosphate; D011493:Protein Kinase C; D000251:Adenosine Triphosphatases; D009218:Myosins; D005033:Ethylmaleimide