C.P.18 Skeletal muscle myopathy mutations at the actin tropomyosin interface that cause gain or loss of function

• Published in: Neuromuscular disorders : NMD Vol. 22; no. 9; p. 846
• Main Authors: Memo, M, Clarke, N.F, Laing, N.G, Nowak, K.J, Ravenscroft, G, Sewry, C, Jain, R, Jayawant, S, Lehtokari, V.L, Marttila, M, Wallgren-Pettersson, C, Marston, S.B
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.10.2012
• Subjects: Actin; Baculovirus; Biopsy; Calcium; Calcium-binding protein; Filaments; Motility; Muscle contraction; Mutation; Myopathy; Neurology; neuromuscular system; Skeletal muscle; Tropomyosin; Troponin
• ISSN: 0960-8966; 1873-2364
• DOI: 10.1016/j.nmd.2012.06.146

Abstract Two mutations, skeletal α-actin K326N and β-tropomyosin ΔK7 were reported in patients with ‘stiff’ muscles and spontaneous contractures, respectively, suggesting a hypercontractile phenotype. K326N actin was isolated from a patient biopsy and ΔK7 β-tropomyosin was expressed in baculovirus/sf9 cells. Ca2+ regulation of reconstituted thin filaments was studied by quantitative in vitro motility assay. Both mutations increased Ca2+-sensitivity (EC50 mut/WT = 0.37 ± 0.05 and 0.45 ± 0.25 respectively). In recent models of actin-α-tropomyosin in the OFF state, both actin K326 and tropomyosin K7 are located in the actin-tropomyosin interface. We hypothesise that the charge change due to the mutation destabilises the OFF state and moves the thin filament equilibrium towards the ON state, thus accounting for the higher Ca2+-sensitivity. We also examined a pair of mutations that cause loss of function. Skeletal α-actin D292V (from a patient biopsy) and β-tropomyosin E117K (expressed in baculovirus/sf9 cells) cause congenital fibre type disproportion (hypotonia and weak contractility with small type 1 muscle fibres but without nemaline bodies). In reconstituted thin filaments β-tropomyosin E117K caused a decrease in Ca2+-sensitivity (EC50 mut/WT = 2.44 ± 0.55). Addition of tropomyosin to actin D292V filaments caused a complete switch-OFF of motility that could not be reversed by troponin at high Ca2+ or even NEM-S-1. Thus congenitally weak muscles correlate with loss of function at the molecular level. These mutations are not at the interface of the OFF state but have an opposite charge change to the gain-of-function mutations and are in a location that could destabilise the ON state, which may account for the loss of function.