EXPRESSION OF A MUTANT MYOSIN LIGHT-CHAIN THAT CANNOT BE PHOSPHORYLATED INCREASES PARACELLULAR PERMEABILITY

A murine leukemia retroviral vector was engineered to contain the DNA encoding either the wild-type, rat aorta 20-kDa myosin light chain (ML C(20)) or a mutant fd rm of MLC(20) in which Thr(18) and Ser(19) were mutated into alanines. These mutations result in a MLC(20) that cannot be phosphorylated by myosin light chain kinase. An Il-amino acid epit ope from c-myc was added to both MLC(20) sequences to facilitate ident ification of these proteins. Madin-Darby canine kidney cells were stab ly transduced, and MLC(20) expression was demonstrated by Western blot analysis using a myc-specific antibody. MLC(20) exchange was demonstr ated by purifying myosin from the transduced cells and repeating the W estern blot analysis. Actin-activated adenosinetriphosphatase assays o n the purified myosins demonstrated similar to 50% decrease in the rat e of ATP hydrolysis by the myosin containing the mutant MLC(20). Trans epithelial electrical resistance was decreased and mannitol flux was i ncreased across monolayers of cells expressing mutant MLC(20). These d ata demonstrate that MLC(20) phosphorylation is involved in regulating paracellular permeability and epithelial barrier function.