Thin filament Ca2+ binding properties and regulatory unit interactions alter kinetics of tension development and relaxation in rabbit skeletal muscle

摘要

The influence of Ca²⁺ binding properties of individual troponin versus cooperative regulatory unit interactions along thin filaments on the rate tension develops and declines was examined in demembranated rabbit psoas fibres and isolated myofibrils. Native skeletal troponin C (sTnC) was replaced with sTnC mutants having altered Ca²⁺ dissociation rates (koff) or with mixtures of sTnC and D28A, D64A sTnC, that does not bind Ca²⁺ at sites I and II (xxsTnC), to reduce near-neighbour regulatory unit (RU) interactions. At saturating Ca²⁺, the rate of tension redevelopment (kTR) was not altered for fibres containing sTnC mutants with decreased koff or mixtures of sTnC:xxsTnC. We examined the influence of koff on maximal activation and relaxation in myofibrils because they allow rapid and large changes in [Ca²⁺]. In myofibrils with M80Q sTnC^F27W (decreased koff), maximal tension, activation rate (kACT), kTR and rates of relaxation were not altered. With I60Q sTnC^F27W (increased koff), maximal tension, kACT and kTR decreased, with no change in relaxation rates. Surprisingly, the duration of the slow phase of relaxation increased or decreased with decreased or increased koff, respectively. For all sTnC reconstitution conditions, Ca²⁺ dependence of kTR in fibres showed Ca²⁺ sensitivity of kTR (pCa50) shifted parallel to tension and low-Ca²⁺kTR was elevated. Together the data suggest the Ca²⁺-dependent rate of tension development and the duration (but not rate) of relaxation can be greatly influenced by koff of sTnC. This influence of sTnC binding kinetics occurs primarily within individual RUs, with only minor contributions of RU interactions at low Ca²⁺.