The indirect flight muscles (IFMs) of Lethocerus (giant water bug) and Drosophila (fruitfly) are asynchronous: oscillatory contractions are produced by periodic stretches in the presence of a Ca2+ concentration that does not fully activate the muscle. The troponin complex on thin filaments regulates contraction in striated muscle. The complex in IFM has subunits that are specific to this muscle type, and stretch activation may act through troponin. Lethocerus and Drosophila have an unusual isoform of the Ca2+-binding subunit of troponin, troponin C (TnC), with a single Ca2+-binding site near the C-terminus (domain IV); this isoform is only in IFMs, together with a minor isoform with an additional Ca2+-binding site in the N-terminal region (domain II). Lethocerus has another TnC isoform in leg muscle which also has two Ca2+-binding sites. Ca2+ binds more strongly to domain IV than to domain II in two-site isoforms. There are four isoforms in Drosophila and Anopheles (malarial mosquito), three of which are also in adult Lethocerus. A larval isoform has not been identified in Lethocerus. Different TnC isoforms are expressed in the embryonic, larval, pupal and adult stages of Drosophila; the expression of the two IFM isoforms is increased in the pupal stage. Immunoelectron microscopy shows the distribution of the major IFM isoform with one Ca2+-binding site is uniform along Lethocerus thin filaments. We suggest that initial activation of IFM is by Ca2+ binding to troponin with the two-site TnC, and full activation is through the action of stretch on the complex with the one-site isoform.
Abbreviations used: AgTnC, Anopheles gambiae TnC (troponin C); DmTnC, Drosophila melanogaster TnC; DsTnC, Drosophila silvestris TnC; E69A mutant (etc.), glutamic acid69 → alanine (etc.); EST, expressed sequence tag; His6, hexahistidine; IFM, indirect flight muscle; LiTnC, Lethocerus indicus TnC; Ni-NTA, Ni2+-nitrilotriacetate; RabTnC, rabbit TnC; RACE, rapid amplification of cDNA ends; Tm, tropomyosin; TnH, troponin H; TnI, troponin I; TnT, troponin T; TEV, tobacco etch virus; WT, wild-type.
Present address: Institut für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum, D-68135 Mannheim, Germany.