Functional Effects of Nemaline Myopathy Mutations on Human Skeletal α-Actin

Abstract

Mutations in human alpha-skeletal actin have been implicated in causing congenital nemaline myopathy, a disease characterized histopathologically by nemaline bodies in skeletal muscle and manifested in the patient as skeletal muscle weakness. Here we investigate the functional effects of three severe nemaline myopathy mutations (V43F, A138P, and R183G) in human alpha-skeletal actin. Wild-type and mutant actins were expressed and purified from the baculovirus/insect cell expression system. The mutations are located in different subdomains of actin; Val-43 is located in a flexible loop of subdomain 2, Ala-138 is near a hydrophobic cleft in the "hinge" region between subdomains 1 and 3, and Arg-183 is near the nucleotide-binding site. None of the three mutations affected the folding of the actin monomer, the velocity at which skeletal myosin moves actin in an in vitro motility assay, or the relative average isometric force supported by F-actin. Defects in fundamental actomyosin interactions are, therefore, unlikely to account for the muscle weakness observed in affected patients. There were, however, significant changes observed in the polymerization kinetics of V43F and A138P and in the rate of nucleotide release for V43F. No detectable defect was found for R183G. If these subtle changes in polymerization observed in vitro are amplified in the context of the sarcomere, it could in principle be one of the primary insults that triggers the development of nemaline myopathy.