Functional delineation of the Ca(2+)-deficient EF-hand in cardiac muscle, with genetically engineered cardiac-skeletal chimeric troponin C.
Cardiac and fast skeletal isoforms of TnC each comprise four putative EF-hand (helix-loop-helix) motifs as potential Ca(2+)-binding sites (sites 1-4), except that site 1 in cardiac TnC is deficient in Ca2+ coordination. In skeletal TnC, the N-terminal sites 1 and 2 are both essential for the trigger mechanism of the contraction switch. However, the mechanism in cardiac muscle is unsettled; it is obscure whether the cardiac site 1 is functionally inert due to calcium deficiency and consequently site 2 is the lone trigger site, or whether sites 1 and 2 perform interactively despite the impairment. These possibilities were addressed by mutagenizing site 1 in skeletal TnC to mimic the cardiac response. In one mutant (STnC-1), two selected Ca(2+)-ligands were abolished. In another (C1/S chimera), 41 N-terminal residues from cardiac TnC were spliced to STnC. The Ca(2+)-binding capacities as well as skinned fiber responses were measured. The STnC-1 derivative failed to switch on contraction. In contrast, the chimeric construct expressed close to full contractile potential in myocardium (74 +/- 3% Po; Po = maximal tension) and also the manifest cardiac phenotype. By devising supplemental chimeric constructs, cardiac-type N-terminal overhang together with cardiac-type EF-hand for site 1 both were found essential for the phenotype. We conclude that cardiac TnC site 1 is actively engaged in the trigger mechanism and in fact dominates the phenotype despite the inability to chelate Ca2+. The N-terminal overhang also participates in this mechanism, which is a novel finding. The conclusion that a non-chelating site functions interactively with a proximal site in cardiac TnC may have wider significance, inasmuch as similar pairings of disparate EF-hands are of common occurrence.
Gulati
J
J
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D005796:Genes; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009132:Muscles / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D015183:Restriction Mapping; D013324:Strontium / Q000494:pharmacology; D014336:Troponin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C
D011993:Recombinant Fusion Proteins; D014336:Troponin; D019209:Troponin C; D002118:Calcium; D013324:Strontium