Phosphorylation of a twitchin-related protein controls catch and calcium sensitivity of force production in invertebrate smooth muscle.
"Catch" is a condition of prolonged, high-force maintenance at resting intracellular Ca2+ concentration ([Ca2+]) and very low energy usage, occurring in invertebrate smooth muscles, including the anterior byssus retractor muscle (ABRM) of Mytilus edulis. Relaxation from catch is rapid on serotonergic nerve stimulation in intact muscles and application of cAMP in permeabilized muscles. This release of catch occurs by protein kinase A-mediated phosphorylation of a high (approximately 600 kDa) molecular mass protein, the regulator of catch. Here, we identify the catch-regulating protein as a homologue of the mini-titin, twitchin, based on (i) a partial cDNA of the purified isolated protein showing 77% amino acid sequence identity to the kinase domain of Aplysia californica twitchin; (ii) a polyclonal antibody to a synthetic peptide in this sequence reacting with the phosphorylated catch-regulating protein band from permeabilized ABRM; and (iii) the similarity of the amino acid composition and molecular weight of the protein to twitchin. In permeabilized ABRM, at all but maximum [Ca2+], phosphorylation of twitchin results in a decreased calcium sensitivity of force production (half-maximum at 2.5 vs. 1.3 microM calcium). At a given submaximal force, with equal numbers of force generators, twitchin phosphorylation increased unloaded shortening velocity approximately 2-fold. These data suggest that aspects of the catch state exist not only at resting [Ca2+], but also at higher submaximal [Ca2+]. The mechanism that gives rise to force maintenance in catch probably operates together, to some extent, with that of cycling myosin crossbridges.
Siegman
M J
MJ
Department of Physiology, Jefferson Medical College, Philadelphia, PA 19107, USA. siegmanm@jeflin.tju.edu
Funabara
D
D
Kinoshita
S
S
Watabe
S
S
Hartshorne
D J
DJ
Butler
T M
TM
D000595:Amino Acid Sequence; D000818:Animals; D049872:Bivalvia; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000378:metabolism* / Q000502:physiology*; D009130:Muscle, Smooth / Q000502:physiology*; D010766:Phosphorylation; D016415:Sequence Alignment
D029742:Caenorhabditis elegans Proteins; D002148:Calmodulin-Binding Proteins; D009124:Muscle Proteins; C489233:unc-22 protein, C elegans; D000242:Cyclic AMP; D002118:Calcium