Srinivasa Narayan

Phosphorylation of a high molecular weight (∼600 kDa) protein regulates catch in invertebrate smooth muscle

A unique property of smooth muscle is its ability to maintain force with a very low expenditure of energy. This characteristic is highly expressed in molluscan smooth muscles, such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis, during a contractile state called ‘catch’. Catch occurs following the initial activation of the muscle, and is characterized by prolonged force maintenance in the face of a low [Ca2+]i, high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage. In the intact muscle, rapid relaxation (release of catch) is initiated by serotonin, and mediated by an increase in cAMP and activation of protein kinase A. We sought to determine which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, having a molecular weight of ∼600 kDa and a molar concentration about 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical conditions studied (rest, activation, catch, and release of catch), changes in phosphorylation of all other proteins, including myosin light chains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the ∼600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorporated per mole of ∼600 kDa protein. Inhibition of A kinase blocked both the cAMP-induced increase in phosphorylation of the protein and the release of catch. In addition, irreversible thiophosphorylation of the protein prevented the development of catch. In intact muscle, the degree of phosphorylation of the protein increases significantly when catch is released with serotonin. In muscles pre-treated with serotonin, a net dephosphorylation of the protein occurs when the muscle is subsequently put into catch. We conclude that the phosphorylation state of the ∼600 kDa protein regulates catch

Regulation of Catch Muscle by Twitchin Phosphorylation: Effects on Force, ATPase, and Shortening

Recent experiments on permeabilized anterior byssus retractor muscle (ABRM) of Mytilus edulis have shown that phosphorylation of twitchin releases catch force at pCa > 8 and decreases force at suprabasal but submaximum [Ca2+]. Twitchin phosphorylation decreases force with no detectable change in ATPase activity, and thus increases the energy cost of force maintenance at subsaturating [Ca2+]. Similarly, twitchin phosphorylation causes no change in unloaded shortening velocity (Vo) at any [Ca2+], but when compared at equal submaximum forces, there is a higher Vo when twitchin is phosphorylated. During calcium activation, the force-maintaining structure controlled by twitchin phosphorylation adjusts to a 30% Lo release to maintain force at the shorter length. The data suggest that during both catch and calcium-mediated submaximum contractions, twitchin phosphorylation removes a structure that maintains force with a very low ATPase, but which can slowly cycle during submaximum calcium activation. A quantitative cross-bridge model of catch is presented that is based on modifications of the Hai and Murphy (1988. Am. J. Physiol. 254:C99-C106) latch bridge model for regulation of mammalian smooth muscle.

The Myosin Cross-Bridge Cycle and Its Control by Twitchin Phosphorylation in Catch Muscle

The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPgammaS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca(2+)] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.

The effect of diazepam on tension and electrolyte distribution in frog muscle

The effect of diazepam on sartorius muscles of the frog was evaluated. Resting tension in sartorius muscle was not affected by diazepam (5 x 10(-5)M) but twitch tension was increased and tetanus tension decreased. The kinetics of 45Ca efflux were altered by diazepam. The calcium content of the intermediate pool was increased by diazepam (5 x 10(-6)M). When the diazepam concentration was increased (5 x 10(-5)M), the time constant of the slow pool decreased and the 45Ca content of the intermediate pool increased further. It is suggested that diazepam interferes with the calcium sequestering system of the sarcoplasmic reticulum (slow pool) and causes an increase of the calcium content of the myofibrillar space (intermediate pool).

The N-terminal Region of Twitchin Binds Thick and Thin Contractile Filaments: REDUNDANT MECHANISMS OF CATCH FORCE MAINTENANCE*

Catch force maintenance in invertebrate smooth muscles is probably mediated by a force-bearing tether other than myosin cross-bridges between thick and thin filaments. The phosphorylation state of the mini-titin twitchin controls catch. The C-terminal phosphorylation site (D2) of twitchin with its flanking Ig domains forms a phosphorylation-sensitive complex with actin and myosin, suggesting that twitchin is the tether (Funabara, D., Osawa, R., Ueda, M., Kanoh, S., Hartshorne, D. J., and Watabe, S. (2009) J. Biol. Chem. 284, 18015–18020). Here we show that a region near the N terminus of twitchin also interacts with thick and thin filaments from Mytilus anterior byssus retractor muscles. Both a recombinant protein, including the D1 and DX phosphorylation sites with flanking 7th and 8th Ig domains, and a protein containing just the linker region bind to thin filaments with about a 1:1 mol ratio to actin and Kd values of 1 and 15 μm, respectively. Both proteins show a decrease in binding when phosphorylated. The unphosphorylated proteins increase force in partially activated permeabilized muscles, suggesting that they are sufficient to tether thick and thin filaments. There are two sites of thin filament interaction in this region because both a 52-residue peptide surrounding the DX site and a 47-residue peptide surrounding the D1 site show phosphorylation-dependent binding to thin filaments. The peptides relax catch force, confirming the regions central role in the mechanism of catch. The multiple sites of thin filament interaction in the N terminus of twitchin in addition to those in the C terminus provide an especially secure and redundant mechanical link between thick and thin filaments in catch.

Cooperative Mechanisms in the Regulation of Smooth Muscle Contraction

In 1930, Bozler reported that smooth muscle was unique in that it could maintain force with a very low energy expenditure (Bozler, 1930). More recent studies have extended this pioneering work to show that the economy of force maintenance of mammalian smooth muscle can vary to a large extent during the course of an isometric contraction (for review see Butler and Siegman, 1985). Crossbridge cycling rate varies independently of the ability to maintain maximum force, and the underlying regulatory mechanisms are not fully understood. It is known that phosphorylation of the 20 kDa light chain of myosin is central to the process of regulation, but details remain elusive. In vitro biochemical experiments have shown that phosphorylation increases the actin-activated myosin ATPase activity (for review see Hartshorne, 1987). To our knowledge, there has been no study showing that contraction of smooth muscle can occur without some increase in myosin light chain phosphorylation. However, high force can be generated with very low degrees of myosin light chain phosphorylation (Moreland and Moreland, 1987; Ratz and Murphy, 1987). Although there are some important exceptions, in general, the maximum velocity of shortening and energy usage are high under conditions when the degree of light chain phosphorylation is high. The major question that really distills from all of the above is what role does myosin light chain phosphorylation play in the regulation of force output and crossbridge cycling rate?