New insights into the functional significance of the acidic region of the unique N-terminal extension of cardiac troponin I

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• 作者：Marcus Henze^a ; ¹ ; Stacey E. Patrick^a ; ¹ ; Aaron Hinken^a ; Sarah B. Scruggs^b ; ^c ; Paul Goldspink^d ; Pieter P. de Tombe^e ; Minae Kobayashi^a ; Peipei Ping^b ; ^c ; Tomoyoshi Kobayashi^a ; R. John Solaro^a ; ^{solarorj@uic.edu}
• 关键词：cTn ; cardiac troponin ; cTnI ; cardiac troponin I ; cTnT ; cardiac troponin T ; cTnC ; cardiac troponin C ; Tm ; tropomyosin ; SwP ; switch peptide ; Ip ; inhibitory peptide ; Wt ; wild type
• 刊名：Biochimica et Biophysica Acta (BBA)/Molecular Cell Research
• 出版年：2013
• 出版时间：April, 2013
• 年：2013
• 卷：1833
• 期：4
• 页码：823-832
• 全文大小：1201 K

文摘

Previous structural studies indicated a special functional role for an acidic region composed of residues 1-10 in the unique N-terminal peptide of cardiac troponin I (cTnI). Employing LC-MS/MS, we determined the presence of phosphorylation sites at S5/S6 in cTnI from wild type mouse hearts as well as in hearts of mice chronically expressing active protein kinase C-¦Å (PKC¦Å) and exhibiting severe dilated cardiomyopathy (DCM). To determine the functional significance of these phosphorylations, we cloned and expressed wild-type cTnI, (Wt), and cTnI variants expressing pseudo-phosphorylation cTnI-(S5D), cTnI(S6D), as well as cTnI(S5A) and cTnI(S6A). We exchanged native Tn of detergent-extracted (skinned) fiber bundles with Tn reconstituted with the variant cTnIs and measured tension and cross-bridge dynamics. Compared to controls, myofilaments controlled by cTnI with pseudo-phosphorylation (S6D) or Ala substitution (S6A) demonstrated a significant depression in maximum tension, ATPase rate, and k_tr, but no change in half-maximally activating Ca^2 +. In contrast, pseudo-phosphorylation at position 5 (S5D) had no effects, although S5A induced an increase in Ca^2 +-sensitivity with no change in maximum tension or k_tr. We further tested the impact of acidic domain modifications on myofilament function in studies examining the effects of cTnI(A2V), a mutation linked to DCM. This mutation significantly altered the inhibitory activity of cTnI as well as cooperativity of activation of myofilament tension, but not when S23/S24 were pseudo-phosphorylated. Our data indicate a new functional and pathological role of amino acid modifications in the N-terminal acidic domain of cTnI that is modified by phosphorylations at cTnI(S23/S24). This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.