Smooth Muscle Myosin Phosphorylated at Single Head Shows Sustained Mechanical Activity

Abstract

Smooth muscle contraction is regulated by the phosphorylation of myosin. It is well known that tonic smooth muscles can maintain force with low energy consumption (latch state); however, the molecular mechanism underlying this phenomenon is unresolved. Here we show that single-head phosphorylated smooth myosin (SHPMII) exhibits fast (ϳ24 s ؊1 ) and slow prolonged (ϳ1 s ؊1 ) actin interactions, whereas double-head phosphorylated myosin (DHPMII) predominantly exhibits the fast (ϳ29 s ؊1 ) interaction, suggesting that the phosphorylated head of SHPMII is mechanically as active as that of DHPMII. Both the fast and the slow actin interactions of SHPMII support the positive net mechanical displacement of actin. The actin translocating velocity of SHPMII was much slower than that of DHPMII, which is consistent with the slow actin interaction of SHPMII. We propose that the "latch state" can be explained by the motor characteristics of SHPMII that is present during the sustained phase of contraction.Smooth muscle is distributed in a variety of tissues such as airways, vasculature, digestive tract, bladder, and uterus. Some smooth muscle cells, such as found in digestive tract, ileum, bladder, and uterus, are mainly relaxed and show phasic contractions upon stimulations. These smooth muscles (often referred to as "phasic smooth muscles") contract and relax rapidly, and their phasic responses are advantageous to the physiological functions such as peristalsis. On the other hand, other smooth muscle cells, such as the smooth muscle cells in airways and vasculature, show sustained contractions, and thus they are often referred to as "tonic smooth muscles." Tonic smooth muscles contract slowly, and very importantly, they can maintain force with very low energy consumption (1, 2). This state of tonic contraction is referred to as the "latch state," and the tone of the contraction plays critical roles in the change in dimension of the hollow organs for regulating airflow, blood flow, organ perfusion, filtration rate of glomeruli, and so forth. Dysregulation of these smooth muscles causes a variety of pathophysiological states such as asthma and hypertension.Contractions of both phasic and tonic smooth muscles are regulated by the phosphorylation of the regulatory light chain (RLC) 3 of smooth muscle myosin (3-8), and the muscle shortening velocity is closely correlated with the extent of the phosphorylation. Upon agonist stimulations, both phasic and tonic smooth muscles show a transient increase in force that is closely correlated with the transient increment of myoplasmic Ca 2ϩ concentration and the concomitant RLC phosphorylation. In a phasic contraction, the force returns quickly to the basal level because of the reduction of the myoplasmic Ca 2ϩ concentration (thus the phosphorylation level of RLC) to the basal level. In a tonic contraction, however, although the level of myosin phosphorylation declines after the transient increment of the myoplasmic Ca 2ϩ concentration, it does not completely return to the basal level, an...

Smooth Muscle Heavy Meromyosin Phosphorylated on One of Its Two Heads Supports Force and Motion

“…Conclusions-The present results agree with previous studies (19,20) in that single-phosphorylated smooth muscle myosin and HMM molecules are active species. This result is relevant for smooth muscle physiology in that single-phosphorylated myosin must exist during activation and, more importantly, during sustained periods of force maintenance.…”

“…It is possible that the biochemical conditions used by Tanaka et al (20) to exchange RLC in their preparation may have resulted in a small fraction of modified, unregulated myosin capable of binding actin. If so, this might explain their observation that single-phosphorylated myosin has both short lifetimes characteristic of their double-phosphorylated species and a fraction of long lifetimes equivalent to their unphosphorylated myosin (20). The molecular mechanics of their single-phosphorylated myosin could sim- ply be a mixture of single-phosphorylated myosin that, as in our study, is indistinguishable from the double-phosphorylated species and a modified unregulated component to account for the long-lived lifetime component.…”

“…It is possible that the methods for preparing HMM-1P contribute to these experimental differences, because our protein expression system does not require the additional biochemical procedures required for RLC removal or exchange (19,20).…”

“…Single-phosphorylated heavy meromyosin (HMM) had much less than half the hydrolytic and mechanical activity of double-phosphorylated HMM when prepared using light chain exchange or stripping protocols (19,20). Using differential tagging of constructs expressed in Sf9 cells followed by sequential affinity columns, the single-phosphorylated HMM (HMM-1P) had more than half the ATPase activity and actin filament speeds in the in vitro motility assay that were similar to doublephosphorylated HMM (1).…”

“…Recently, the approach to this problem has been improved by employing various methods that allow isolation of a singlephosphorylated species (1,19,20). Single-phosphorylated heavy meromyosin (HMM) had much less than half the hydrolytic and mechanical activity of double-phosphorylated HMM when prepared using light chain exchange or stripping protocols (19,20).…”