Troponin Isoforms and Stretch-activation of Insect Flight Muscle

Abstract

Oscillatory contraction of insect indirect flight muscle (IFM) is activated by sinusoidal length changes. Work done by oscillating fibres is measured from the area of loops on a length-tension plot. At [Ca2+] above 10 μM, progressively less oscillatory work is produced because fibres contract isometrically and are unable to relax fully after each cycle of oscillation. Periodic stretches during oscillations activate fibres through the action of TnC F1, which binds one Ca2+ in the C-lobe. Activation of isometric contraction by Ca2+ acts through F2, which binds Ca2+ in both N- and C-lobes. Lethocerus IFM fibres substituted with F1gave oscillatory work, which did not decline at high [Ca2+], while fibres substituted with F2 produced more isometric tension as [Ca2+] was increased. Varying proportions of F1 and F2 gave maximal work with an F1:F2 ratio of 100:1, which is higher than the in vivo ratio of 7:1. The structure of F1, and the interaction with TnI, were determined by NMR. The N-lobe of F1 is in the closed conformation in apo and Ca2+- bound forms and does not bind TnI. Unexpectedly, the C-lobe is open in both states, and binds the N-terminal domain of TnI independently of Ca2+. The affinity of F1and F2 for a complex containing tropomyosin, TnT and TnH (Lethocerus TnI) were measured by isothermal calorimetry in the presence of Ca2+. The affinities of F1 and F2 for the complex were 5.4 μm and 65 nM respectively. This difference is likely to be due to a single TnI binding site on F1 and two sites on F2. Stretch may be sensed by an extended C-terminal domain of TnH, and transmitted to the C-lobe of F1, resulting in a change in the interaction of the TnI inhibitory domain and actin.