Binding properties of the calcium-activated F2 isoform of Lethocerus troponin C.
While in most muscles contraction is triggered by calcium effluxes, insect flight muscles are also activated by mechanical stretch. We are interested in understanding the role that the troponin C protein, usually the calcium sensor, plays in stretch activation. In the flight muscles of Lethocerus, a giant water bug often used as a model system, there are two isoforms of TnC, F1 and F2, present in an approximately 10:1 ratio. F1 TnC is responsible for activating the muscle following a stretch, whereas F2 TnC produces a sustained contraction, the magnitude of which depends on the concentration of Ca(2+) in the fiber. We have previously shown that F1 TnC binds only one Ca(2+) ion in its C-terminal domain and that interaction with troponin H, the insect ortholog of troponin I, is insensitive to Ca(2+). Here, we have studied the effect of Ca(2+) and Mg(2+) on the affinities of the interaction of F2 TnC with troponin H peptides. We show that the presence of two Ca(2+) ions, one in each of the globular domains, increases the affinity for TnH by at least 1 order of magnitude. The N lobe has a lower affinity for Ca(2+), but it is also sensitive to Mg(2+). The C lobe is insensitive to Mg(2+) as previously demonstrated by mutations of the individual EF-hands. The interaction with TnH seems also to have significant structural differences from that observed for the F1 TnC isoform. We discuss how our findings could account for stretch activation.
National Institute for Medical Research, The Ridgeway, London NW71AA, U.K.
Stephen R
SR
Martin
Giovanna
G
Avella
Miquel
M
Adrover
Gian Felice
GF
de Nicola
Belinda
B
Bullard
Annalisa
A
Pastore
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020062:Heteroptera; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism*; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism*
D019476:Insect Proteins; D020033:Protein Isoforms; D019209:Troponin C; D002118:Calcium
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