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

Abstract

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.

Twitchin from Molluscan Catch Muscle

“…Based on a partial cDNA of the purified isolated protein, it was found to be a homologue of the mini-titin, twitchin (11), that is associated with the thick filament (12). The mechanism by which twitchin controls force output seems to reside in the ability of unphosphorylated twitchin to slow the detachment of calcium-free myosin from actin, primarily by slowing the rate of ADP release from attached cross-bridges (13,14). In the presence of phosphorylated twitchin, crossbridge detachment is fast at low intracellular [Ca 2ϩ ] (14).…”

“…The mechanism by which twitchin controls force output seems to reside in the ability of unphosphorylated twitchin to slow the detachment of calcium-free myosin from actin, primarily by slowing the rate of ADP release from attached cross-bridges (13,14). In the presence of phosphorylated twitchin, crossbridge detachment is fast at low intracellular [Ca 2ϩ ] (14). In vitro studies on both native and reconstituted thick filaments from Mytilus also show the primary role that twitchin and its phosphorylation state play in regulation of the catch state (15).…”

Molecular Motors: Force and Movement Generated by Single Myosin II Molecules

“…Indeed, there is evidence (reported in Ref. 2) suggesting that, like the latch, the catch might well be due to the persistence of a locked actomyosin-ADP state or, for that matter, an intermediate attached state similar to that observed in myosin I. Thus actively contracting catch muscle may enter the catch state if its cycling cross bridges are trapped at low free calcium levels in a long-lived actomyosin-ADP state, stabilized perhaps by the high tension that pulls on the attached cross bridge.…”

“…Recently Butler and colleagues (2) proposed that a longlived intermediate attached state of myosin (i.e., an actomyosin-ADP state) may also account for the so-called catch phenomenon observed in certain molluscan smooth muscle such as the adductor muscles of bivalves, e.g., the scallop or the oyster, that may keep the shells closed for many hours and even days apparently without fatigue and barely any oxygen consumption. Another catch muscle, the anterior byssus retractor muscle of the edible mussel Mytilus edulis, develops an enormous tension (up to 15 kg/cm 2 cross section) when stimulated electrically or with acetylcholine but relaxes extremely slowly or even fails to completely relax after cessation of stimulation.…”

“…Furthermore, like the latch, the catch state too may be terminated by phosphorylation of a protein associated with the myosin-containing myofilaments. However, the myosinbound protein concerned is twitchin (a kind of mini-titin) rather than the myosin light chains that are involved in the regulation of the latch state of vertebrate smooth muscle (2). Physiologically, this phosphorylation is brought about by protein kinase A, for instance in an oyster when its adductor muscles are stimulated by the neurotransmitter serotonin liberated from inhibitory nerves that eventually cause the shells to open.…”