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Lecture 12

BIOL 4510 Lecture 12-contractile proteins 2-revised.pdf

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BIOL 4510
Peter Backxx

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BIOL4510 / KINE 4510 Contractile Proteins 2 Peter Backx, 2012 General Discussion of Force generation: Force generation involves the interaction of thick and thin filaments. In skeletal and cardiac muscle this is controlled by Ca 2+ binding to the troponin-tropomyosin complex 2+ located on the thin filament. The regulation of force generation by Ca 2+s highly cooperative which allows force to be turned on over a very narrow range of Ca levels. For smooth muscle the force is regulated by phosphorylation of the regulatory MLC-2 by myosin-light-chain kinase, which is activated by a number of signaling pathways, many 2+ 2+ of which are activated by Ca so there is still Ca regulation. Some muscle videos to watch Introduction to contraction: Process of (skeletal muscle excitation Cross-bridge cycle: view these two videos together. 1 Thin Filament Proteins Tropomyosin-Troponin Complex Troponin-tropomyosin complex 2+ -site of (most of) Ca regulation of contraction in heart and skeletal muscle Tropomyosin = Tm (MW 42,000) - 4 human tropomyosin genes: TPM1 (), TPM2 (), TPM3 (slow ) and TPM4 - in striated muscle (skeletal and cardiac) muscle, the principal Tms are and - in heart: small mammals (rat, guinea pig, rabbit): exclusively larger mammals (human, sheep, pig, cow) = 5:1 - Fibrous protein formed by dimerization of two tropomyosins: form -helical coil- coil structure with length ~38-40nm which is just slightly longer than the thin filament repeat (36nm) - These dimers lie in groove formed by filamentous actin (1 dimer in each groove) - each tropomyosin dimer molecule spans 7 actin monomers: therefore 2 Tm dimers per 7 actins (1 in each groove) - Tms interaction end-to-end along the actin filament which is responsible for much (but not all) of the "co-operativity of Ca2+binding and force generation" discussed (see below) - Tm has 7 internal repeat domains, each containing a single actin binding site - in low Ca 2+ (i.e. relaxed non-force generating), tropomyosin sits in a position that prevents myosin cross bridges from binding actin - Tm covers 3 of the 4 myosin binding sites located on each actin monomer (when myosin binds to actin there are 4 sites of contact and interaction) 2+ - position of the tropomyosin on the thin filament changes when Ca binds to troponin-C (as well as with cross-bridge attachment). Tropomyosin moves further into the groove and exposes the myosin binding sites on actin. This movement is transmitted by changes in the Troponin complex which occurs simultaneously with 2+ 2+ Ca binding to TnC. In other words, the energy of Ca binding is coupled to conformational changes in the troponin-tropmyosin complex leading to the movement of tropomyosin away from the myosin binding sites on the actin monomers. 2 FIGURE Tropomyosin structure. Tropomyosin (Tm) lies in the grooves formed by the coiled-coiled filamentous actin. Tropomyosin complexes with other thin filament Troponin proteins (troponin-C, troponin-T, troponiPosition of tropomyosin on the thin filament changes whenis elevated due to Ca -dependent conformation changes that occur in the Troponin complex when Ca binds to Troponin-C. Troponin complex = 3 proteins bound together = 1 Troponin-T (Tn-T; MW 38,000; tropomyosin binding Troponin protein), 1 Tn-C (MW 18,000; calcium binding protein) + 1 Tn-I (MW 23,000; inhibitory unit which binds to actin). Tn-T: Three genes: TNNT1 slow skeletal muscle TNNT2 cardiac (cTnT), 4 splice variants isoforms (cTnT1, TnT2, cTnT3, cTnT4) TNNT3 fast skeletal muscle - two major isoforms of cardiac Tn-T (TNNT2): and MW: - Tn-T forms dimers of and attached by disulfide bridge - One of the most charged proteins in the body (30% acidic, 20% basic residues). - molecule is long = lies along Tm for about 3 actin monomers - Tn-T glues the troponin complex to Tm and thereby prevents myosin from binding to actin - Tn-T binds Tm via its N-terminus which sits near and overlays the Tm-Tm interaction regions (involved in cooperativity) - C-terminus of Tn-T is globular and bind to Tn-C and Tn-I FIGURE: Structure of Tn-T 3 - Tn-T has a globular C-terminus that interacts with Tn-C and Tn-I - Tn-T also enhances the myosin ATPase once the myofilaments are activated 2+ - Ca binding to Tn-C (see below) strengthens interactions between Tn-T and Tm Additional clinical facts about TnT: - mutations of Tn-T are linked to cardiomyopathy; many of these cardiomyopathies have a distinct clinical presentation characterized by very poor diastolic function (hearts do not relax). What impact will this have on the ability of the heart to pump blood (think about the P-V representation of the cardiac cycle)? - Tn-T is proteolyzed during ischemia-reperfusion and used as a marker for heart damage 2+ - Tn-T is phosphorylate2+by PKC (not PKA): phosphorylation reduces maximal Ca - activated force, Ca sensitivity and the cross-bridge cycling rate Tn-C: - two genes TNNC1 cardiac and slow skeletal muscle isoform (cTnC) TNNC2 f2+t skeletal muscle isoform (sTnC) - responsible for Ca sensitivity of force generation - cardiac Tn-C can bind 3 Ca 2+ ions/molecule but only one is specific for Ca 2+ under physiological conditions 2+ 2+ - 2 non-specific Ca /Mg binding sites (sites III and IV): these sites are required for structural integrity - 1 Ca specific high affinity site (site II): the KD for binding is about 1.7 m 2+ 2+ 2+ - site I in cTnC does not bind Ca or Mg . Therefore, only one Ca ion needs to bind to cTnC to turn on myosin actin interactions in the 7 actin monomers regulated by a cTnC protein - MW of cTnC similar to calmodulin: an ubiquitous molecule involved in many Ca - 2+ mediated transduction mechanisms in cells (activation of calcineurin, activation of 2+ muscle contraction in smooth muscle cells (later), inactivation of L-type Ca channels etc etc) - skeletal muscle sTn-C has 2 Ca specific sites/low affinity site (i.e. sites I and II). 2+ So 2 Ca must bind to sTnC to initiate myosin-actin interactions. 4
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