Expression of a mutant myosin light chain that cannot be
phosphorylated increases paracellular permeability.
Gandhi, Sunil, Donald D. Lorimer, and Primal De Lanerolle.
Departments of Physiology and Biophysics and Surgery, University of
Illinois at Chicago, Chicago, IL 60612
APStracts 3:0194F, 1996.
A murine leukemia retroviral vector was engineered to contain the DNA
encoding either the wild type, rat aorta 20 kDa myosin light chain
(MLC20) or a mutant form of MLC20 in which thr 18 and ser 19 were
mutated into alanines. These mutations result in a MLC20 that cannot
be phosphorylated by myosin light chain kinase. An 11 amino acid
epitope from c-myc was added to both MLC20 sequences to facilitate
identification of these proteins. Madin-Darby canine kidney cells
were stably transduced and MLC20 expression was demonstrated by
Western blot analysis using a myc-specific antibody. MLC20 exchange
was demonstrated by purifying myosin from the transduced cells and
repeating the Western blot analysis. Actin-activated ATPase assays on
the purified myosins demonstrated 50% decrease in the rate of ATP
hydrolysis by the myosin containing the mutant MLC20. Transepithelial
electrical resistance was decreased and mannitol flux was increased
across monolayers of cells expressing mutant MLC20. These data
demonstrate that MLC20 phosphorylation is involved in regulating
paracellular permeability and epithelial barrier function.
Received 13 February 1996; accepted in final form 17 October
1996.
APS Manuscript Number F47-6.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996