Comparison of the Binding of the Switch Regions of Cardiac Troponin-I and Skeletal Troponin-I to the Functional N-Domain of Human Cardiac Troponin-C

Abstract

In the heart, there are two isoforms of troponin-I (TnI) that are developmentally regulated. The slow skeletal TnI (ssTnI) is the sole isoform expressed in the embryonic or neonatal heart, while cardiac TnI (cTnI) is expressed exclusively in the adult heart. One important distinction between ssTnI and cTnI relates to differences in myofilament Ca2+-sensitivity of force development under acidic pH conditions. Hearts expressing ssTnI show heightened Ca2+-sensitivity compared with hearts expressing cTnI under basal conditions. This isoform-specific difference is greatly accentuated during acidosis, which is often associated with ischemia of myocardial infarction. In vitro and in vivo functional studies have shown that replacement of cTnI with ssTnI results in a marked enhancement of myofilament Ca2+-sensitivity at acidic pH conditions. Recent reports have indicated that this effect can be ascribed to amino acid sequence differences in ssTnI and cTnI and in particular to a critical A162H substitution in the switch region. In this study, we have used NMR spectroscopy to examine the binding of the switch regions of ssTnI (sTnI115-131) and cTnI (cTnI147-163) to the N-domain of cardiac troponin-C (cNTnC) at physiological and acidic pH conditions. The results show that the affinity of sTnI115-131 for cNTnC•Ca2+ (KD ∼50uM) is ∼3-fold stronger than that of cTnI147-163 (KD ∼150uM), but neither are affected by a pH change from 7 to 6. The pKa of H130 in sTnI115-131 is 6.2 when free and 6.7 when bound to cNTnC•Ca2+. We have also used {1H, 15N}-HSQC NMR spectroscopy to monitor the pKa changes of cNTnC•Ca2+ from peptide free to peptide bound states. The implications of these results will be discussed in the context of structure and function of myofilament protein interactions.