Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin-I mutants and troponin-C

Troponin C (TnC) could be extracted from skinned porcine cardiac muscle fibres and their Ca2+ sensitivity restored by reconstitution with recombinant human cardiac TnC, After extraction of troponin I (TnI) and TnC using the vanadate treatment method of Strauss et al, [Strauss, J. D., Zeugner, C., Van Eyk, J.E., Bletz, C., Troschka, M. and Ruegg, J.C. (1992) FEES Lett. 310, 229-234], skinned porcine cardiac muscle fibres were reconstituted with wild-type recombinant human cardiac TnC and either wild-type cardiac TnI or several mutant isoforms of human TnI, Reconstitution with mild-type proteins restored the Ca2+ sensitivity of the tissue and phosphorylation of the TnI with the catalytic subunit of protein kinase A reduced the Ca2+ sensitivity (i.e. -log[Ca2+] for 50% of maximal force) as has been shown by others, However, reconstitution with the TnI mutant Ser-23Asp/ Ser-24Asp mimicking the phosphorylated form of cardiac TnI, led to a reduced Ca2+ sensitivity compared with reconstitution with wild-type TnI, whereas the mutant Ser-23Ala/Ser-24Ala behaved as the dephosphorylated form of TnI, These data confirm the importance of negative charge in this region of the TnI molecule in altering the Ca2+ responsiveness in this system.