highly penetrant point mutations located within the cTnT N-terminus (R92L/W and I79N) which are associated with distinct phenotypes and severities. We employed stopped-flow to measure the rates of calcium association (kon) to and dissociation (koff) from IAANS-labeled cardiac troponin C in CTF systems of increasing complexity to explain similar changes in steady-state calcium sensitivity often associated with these mutations. We previously characterized mutation-specific differences in koff for these mutations in CTFs. Kon to WT, R92L, R92W, and I79N CTFs was measured over a range of calcium concentrations and calculated to be 9.8852.051x106M-1s-1, 9.14151.94x106M-1s-1, 31.3752.948x106M-1s-1, and 28.252.736x106M-1s-1, respectively. Both I79N and R92W, but not R92L, significantly increased kon compared to WT CTFs. We then explored the effects of these mutations on koff in the presence of heavy meromyosin (HMM). Koff from WT, R92L, R92W, and I79N CTFsþHMM was found to be 12.9850.170s-1, 10.0850.147s-1, 7.8250.267s-1, and 9.6950.354s-1, respectively. After adding HMM to the system, all three mutations significantly decreased koff to a similar extent (1.3-fold). Lastly, we added C0C2, a cardiac myosin binding protein-C fragment, to the WT CTF system. Adding C0C2 decreased koff in a concentrationdependent manner that was abolished upon C0C2 phosphorylation. These results highlight the utility of stopped-flow for probing myofilament calcium kinetics in vitro and how mutation-specific alterations in calcium kinetics can be used to distinguish mutations which similarly affect calcium sensitivity.
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