Central helix role in the contraction-relaxation switching mechanisms of permeabilized skeletal and smooth muscles with genetic manipulation of calmodulin.
A prominent common feature of calmodulin and troponin structures is the unusually long central helix which separates the two lobes, each containing two Ca2(+)-binding sites. To study the role of certain highly conserved residues in the helix in the contraction-relaxation switching mechanism in muscle, we measured the Ca2(+)-activated force of permeabilized skeletal and smooth muscles with three genetically manipulated forms of calmodulin. Mutated calmodulin was made to substitute for troponin-C in vertebrate skeletal fiber. The mutants had 1-4 deletions in the conserved cluster (positions 81-84) in the solvent-exposed region of the central helix, which also substantially shortened the helix. The force of the maximally activated fiber was found to be diminished only with the mutant in which the entire cluster Ser-81 to Glu-84 (CaM delta 81-84) was deleted. All such deletions were found to be completely ineffective in blocking the Ca2(+)-switching process in smooth muscle strips. The results show for the first time that at least a part of the highly conserved four-residue cluster in the central helix is critical for the contraction mechanism of striated muscle. Further, the possibility is raised that the reduced length of the central helix may be a determining factor in the Ca2(+)-switching mechanism in fast-twitch muscle. These findings combined with the results on smooth muscle indicate diversity in the structure-function specifications for the central helix of calmodulin for different target proteins.
Gulati
J
J
Albert Einstein College of Medicine, Department of Medicine, Bronx, NY 10461.
Persechini
A
A
Babu
A
A
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D066298:In Vitro Techniques; D009119:Muscle Contraction / Q000502:physiology*; D009126:Muscle Relaxation / Q000502:physiology; D009130:Muscle, Smooth / Q000378:metabolism* / Q000502:physiology; D009132:Muscles / Q000378:metabolism*; D010539:Permeability; D011817:Rabbits; D013329:Structure-Activity Relationship; D014336:Troponin / Q000378:metabolism; D019209:Troponin C
D002147:Calmodulin; D014336:Troponin; D019209:Troponin C; D002118:Calcium