A primary mechanism for initiating smooth muscle contraction involves an increase in [Ca 2ϩ ] i , leading to myosin regulatory light chain (RLC) phosphorylation, crossbridge cycling, and force development (1, 2). Phosphorylation of Ser-19 on RLC of myosin II changes the orientation of myosin crossbridges, allowing actin activation of myosin ATPase activity. A similar mechanism occurs with nonmuscle myosin II with effects on many cellular actomyosin-dependent functions. The Ca 2ϩ -dependent phosphorylation of RLC is mediated by Ca 2ϩ ͞cal-modulin (CaM)-dependent myosin light chain kinase (MLCK), whereas myosin light chain phosphatase dephosphorylates RLC. In smooth muscles, agonists stimulate greater RLC phosphorylation and force than do depolarizing stimuli at comparable [Ca 2ϩ ] i because of Ca We developed a different CaM-sensor MLCK capable of monitoring MLCK activation to obtain temporal and quantitative information on Ca 2ϩ ͞CaM binding to MLCK where Ca 2ϩ -dependent CaM binding increased kinase activity coincident with a decrease in FRET (11). The CaM-sensor MLCK was expressed in smooth muscle tissues of transgenic mice to obtain quantitative insights on CaM activation of MLCK relative to [Ca 2ϩ ] i and RLC phosphorylation and force development. These results show that genetically encoded biosensors may be used to investigate physiological processes in tissues of transgenic mice. MethodsConstruction of SM8 35 KCS Plasmid. The pSM8 35 KCS construct was prepared by subcloning the 1.6-kb cDNA of Ca 2ϩ ͞CaM-sensor containing the MLCK CaM-binding sequence flanked by enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) (12) into the pSM8-CAT vector, which contains the smooth muscle ␣-actin promoter (13, 14). The 3.1-kb cDNA fragment of short rabbit smooth muscle MLCK was further subcloned into the site between the Ca 2ϩ ͞ CaM-sensor gene and the smooth muscle ␣-actin promoter in pSM8-CAT vector to produce pSM8 35 KCS construct. Correct This paper was submitted directly (Track II) to the PNAS office.

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Atypical antipsychotic (AA) medications including risperidone (RIS) and olanzapine (OLAN) are FDA approved for the treatment of psychiatric disorders including schizophrenia, bipolar disorder and depression. Clinical side effects of AA medications include obesity, insulin resistance, dyslipidemia, hypertension and increased cardiovascular disease risk. Despite the known pharmacology of these AA medications, however, the mechanisms contributing to adverse metabolic side-effects are not well understood. To evaluate drug-associated effects on the heart, we assessed changes in the cardiac proteomic signature in mice administered for 4 weeks with clinically relevant exposure of RIS or OLAN. Using proteomic and gene enrichment analysis, we identified differentially expressed (DE) proteins in both RIS-and OLAN-treated mouse hearts (p <0.05), including proteins comprising mitochondrial respiratory complex I and pathways involved

A novel translocation step is inferred from structural studies of the interactions between the intracellular calcium receptor protein calmodulin (CaM) and one of its regulatory targets. A mutant of CaM missing residues 2-8 (⌬NCaM) binds skeletal muscle myosin light chain kinase with high affinity but fails to activate catalysis. Small angle x-ray scattering data reveal that ⌬NCaM occupies a position near the catalytic cleft in its complex with the kinase, whereas the native protein translocates to a position near the C-terminal end of the catalytic core. Thus, CaM residues 2-8 appear to facilitate movement of bound CaM away from the vicinity of the catalytic cleft.