Analysis of physiological roles of Drosophila calmodulin through in vivo genetic and in vitro structure/function studies.

摘要

Calmodulin (CaM), a small protein found in all eukaryotes examined, is a major component of Ca2+ signaling pathways and functions as a Ca2+ signal sensor and transducer. A wide variety of targets are regulated by CaM, including enzymes, cytoskeleton elements and ion channels.; To dissect the in vivo roles of Drosophila CaM, a series of Cam mutations were previously generated in the Beckingham lab. This thesis primarily concerns investigation of two Cam mutations; Cam7, a point mutation encoding V91G mutant CaM, and a null mutation, Camn339. Cam7 causes unprecedented defects. Cam 7 mutants are sluggish as larvae and form aberrant pupal cases with highly indented rings around the body. Mutant pupae never eclose and most die as pharate adults with head defects.; Expression of wild type CaM specifically in the musculature is shown here to partially rescue the Cam7 phenotype, suggesting that muscle function is primarily affected by this mutation. Further, genetic studies performed suggest that misregulation of the ryanodine receptor (RyR), a Ca2+ channel on the sarcoplasmic reticulum, may be largely responsible for the muscle defects. Muscle contraction-associated Ca2+ release is shown here to be drastically altered in the Cam7 mutant.; Biochemical studies revealed that the V91G mutation has no detectable effects on Ca2+-binding or the Ca2+-induced conformation of CaM. However, the conformation of Ca2+-free CaM is altered. Examination of the interaction between CaM and the RyR CaM binding region indicates that the V91G mutation would alter regulation of the RyR so as to cause Ca2+ leaking through the RyR channel.; The ability of CaM variants with the N- or C-terminal Ca 2+-binding sites inactivated to rescue the Cam7 phenotype was also investigated. Consistent with prior CaM-RyR studies, the C-terminal binding site mutant exacerbated the phenotype. However, the N-terminal binding site mutant showed an “over-rescue” effect causing muscle relaxation.; In contrast, the main behavioral defect observed in Cam null mutant larvae was found to be rescued by neural, not muscle, expression of wild type CaM and neither the N-terminal nor C-terminal Ca2+-binding site mutants had any effect on this defective behavior, suggesting a different molecular pathway is affected.