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 Rüegg, J.C. (1992) FEBS 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 wild-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.
Dohet
C
C
II Physiologisches Institut, Universität Heidelberg, Germany.
al-Hillawi
E
E
Trayer
I P
IP
Rüegg
J C
JC
D000409:Alanine / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004563:Electrochemistry; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D016658:Psoas Muscles / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine; D014336:Troponin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
D011993:Recombinant Fusion Proteins; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D012694:Serine; D000409:Alanine; D002118:Calcium