Effects of two hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on Ca2+ regulation of thin filament motility.
The functional properties of wild type alpha-tropomyosin expressed in E. coli with an alanine-serine N-terminal leader (AS-alpha-Tm) were compared with those of AS-alpha-Tm with either of two missense mutations (Asp175Asn and Glu180Gly) shown to cause familial hypertrophic cardiomyopathy (FHC). Wild type AS-alpha-Tm and AS-alpha-Tm(Asp175Asn) binding to actin was indistinguishable from rabbit skeletal muscle ab-tropomyosin whilst the affinity of AS-alpha-Tm(Glu180Gly) was about threefold weaker. In vitro motility assays were performed with AS-alpha-tropomyosin incorporated into skeletal muscle actin-rhodamine phalloidin filaments moving over skeletal muscle heavy meromyosin. Under relaxing conditions (pCa9), troponin added to actin filaments containing AS-alpha-tropomyosin or mutant tropomyosins resulted in normal switch-off, with a decrease in the fraction filaments moving from >80% to <20%. Under activating conditions (pCa5), troponin had a minor effect upon actin-AS-alpha-tropomyosin filament velocity (increased by 5 +/- 1%, n=10), whereas the velocity increased by 18 +/- 3% (n=7) with actin filaments containing AS-alpha-tropomyosin(Asp175Asn) and by 21 +/- 2% (n=8) with filaments containing AS-alpha-tropomyosin(Glu180Gly) (p<0.05 compared with AS-alpha-tropomyosin). Thus FHC mutations in alpha-tropomyosin produce detectable changes in the Ca2+-regulation of thin filaments, presumably via altered interaction with troponin.
Bing
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Cardiac Medicine, Imperial College School of Medicine, National Heart and Lung Institute, London, United Kingdom.
Redwood
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Purcell
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Esposito
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Watkins
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Marston
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D000199:Actins / Q000378:metabolism; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid / Q000235:genetics*; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics*; D018698:Glutamic Acid / Q000235:genetics*; D005998:Glycine; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D015879:Myosin Subfragments / Q000378:metabolism; D010590:Phalloidine / Q000378:metabolism; D011817:Rabbits; D012235:Rhodamines / Q000378:metabolism; D014335:Tropomyosin / Q000235:genetics* / Q000502:physiology*; D014336:Troponin / Q000494:pharmacology
D000199:Actins; D015879:Myosin Subfragments; D012235:Rhodamines; D014335:Tropomyosin; D014336:Troponin; D010590:Phalloidine; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D005998:Glycine