Key Points
- To determine whether either of the low-affinity calcium-binding sites in cardiac troponin C can trigger contraction in slow-twitch skeletal muscle fibers.
- Generated two cardiac troponin C mutants via site-directed mutagenesis: one restoring calcium binding to dormant site I (CBM+I) and another activating site I while inactivating site II (CBM+I-IIA).
- Assembled mutant proteins with troponin I and troponin T into troponin complexes and reconstituted them into troponin C-depleted, permeabilized slow-twitch skeletal muscle fibers.
- Assessed protein structural conformations via NMR spectroscopy and evaluated mechanical force generation in response to calcium and strontium.
- Mutant CBM+I supported robust force generation in skinned slow muscle fibers, exhibiting strontium and calcium sensitivities comparable to fast skeletal troponin C.
- Mutant CBM+I-IIA completely failed to restore calcium-dependent contraction in troponin C-depleted skinned muscle fibers.
- NMR spectra confirmed marked structural differences between CBM+I and CBM+I-IIA in the presence of calcium, establishing that low-affinity sites I and II possess non-redundant, distinct functions.
Structured PICO
PPopulationPermeabilized slow-twitch skeletal muscle fibers and engineered cardiac troponin C (TnC) mutants
IInterventionMutagenesis of cardiac TnC to restore dormant low-affinity site I (CBM+I) or activate site I while inactivating site II (CBM+I-IIA)
OOutcomeForce generation and Ca2(+)-dependent contraction in skinned slow skeletal muscle fiberssurrogate
The study demonstrates that low-affinity Ca2+-binding sites I and II in cardiac troponin C have distinct functions, and only site II can trigger muscle contraction.