The role of the Ca2+ regulatory sites of skeletal troponin C in modulatingmuscle fibre reactivity to the Ca2+ sensitizer bepridil

1 The Ca2+-sensor protein troponin C (TnC) exerts a key role in the regulat ion of muscle contraction, and constitutes a target for Ca2+ sensitizer com pounds, such as bepridil, known to increase its apparent Ca2+ affinity. Mor eover, bepridil has been reported to exert a differential effect in slow an d fast skeletal muscle fibres, which express the slow/cardiac and fast TnC isoform, respectively. 2 The role of the TnC isoform in establishing the differential effect of be pridil was assessed in slow soleus and fast tibialis rat skinned fibres, by extraction of endogenous TnC and consecutive reconstitution with either sl ow or fast recombinant TnC. A mutant (VG2), lacking the regulatory site II, was also used to distinguish the role of each regulatory site. 3 Fast tibialis fibres reconstituted with cardiac TnC exhibited a typical s low bepridil reactivity, while slow soleus fibres reincorporated with fast TnC displayed a typically fast reactivity to bepridil. These results indica ted that the differential effect of bepridil in slow and fast fibres is rel ated to the TnC isoform predominantly expressed in a fibre. 4 Experiments with the VG2 mutant demonstrated that BPD can achieve an incr ease in the Ca2+ affinity in the absence of a functional site II. Thus, sit e I was necessary for the BPD effect to be independent of the Ca2+ concentr ation. Moreover, the amplitude of the reinforcement in the Ca2+ affinity, i nduced by the binding of bepridil to the TnC molecule, is dependent on the number of functional regulatory sites, the larger affinity reinforcement be ing detected when only one regulatory site (either site I or II) is functio nal.