Abstract
Molluscan smooth muscles exhibit a low energy cost contraction called catch. Catch is regulated by twitchin phosphorylation and dephosphorylation. Recently, we found that the D2 fragment of twitchin containing the D2 site (Ser-4316) and flanking immunoglobulin motifs (TWD2-S) formed a heterotrimeric complex with myosin and with actin in the region that interacts with myosin loop 2 (Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., and Watabe, S. (2007) J. Exp. Biol. 210, 4399-4410). Here, we show that TWD2-S interacts directly with myosin loop 2 in a phosphorylation-sensitive manner. A synthesized peptide, CAQNKEAETTGTHKKRKSSA, based on the myosin loop 2 sequence (loop 2 peptide), competitively inhibited the formation of the trimeric complex. Isothermal titration calorimetry showed that TWD2-S binds to the loop 2 peptide with a K(a) of (2.44 +/- 0.09) x 10(5) m(-1) with two binding sites. The twitchin-binding peptide of actin, AGFAGDDAP, which also inhibited formation of the trimeric complex, bound to TWD2-S with a K(a) of (5.83 +/- 0.05) x 10(4) m(-1) with two binding sites. The affinity of TWD2-S to actin and myosin was slightly decreased with an increase of pH, but this effect could not account for the marked pH dependence of catch in permeabilized fibers. The complex formation also showed a moderate Ca(2+) sensitivity in that in the presence of Ca(2+) complex formation was reduced.
Highlights
Molluscan smooth muscles, such as mussel anterior byssus retractor muscle (ABRM)2 and adductor muscle, exhibit a low energy cost phase of tension maintenance termed catch
Catch tension is abolished by secretion of serotonin and an increase of intracellular [cAMP] with the resulting activation of cAMP-dependent protein kinase and phosphorylation of twitchin [4, 5]
Twitchin phosphorylation is required for relaxation of the muscle from catch. For this cycle to repeat, dephosphorylation of twitchin is necessary [6]. In this scheme, twitchin is a major regulator of the catch state
Summary
Molluscan smooth muscles, such as mussel anterior byssus retractor muscle (ABRM)2 and adductor muscle, exhibit a low energy cost phase of tension maintenance termed catch. The twitchin-binding peptide of actin, AGFAGDDAP, which inhibited formation of the trimeric complex, bound to TWD2-S with a Ka of (5.83 ؎ 0.05) ؋ 104 M؊1 with two binding sites. We found that a twitchin fragment including the D2 phosphorylation site and its flanking Ig motifs (TWD2-S) interacted with myosin and actin in a phosphorylation-sensitive manner, and it was suggested that this trimeric complex contributed to tension maintenance in catch [11].
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