Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach

Abstract

The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidinetagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain.Keywords: isoforms; mutants; myosin light chains; site-directed mutagenesis; smooth muscle.Myosin II is a major contractile protein composed of two heavy chains (< 200 kDa each) and two pairs of light chains (< 20 kDa each). One pair of light chains is described as regulatory (20 kDa) and the other as essential (17 kDa), based on their distinctive chemical and functional features. In smooth muscle myosin, the two 20-kDa regulatory chains (LC20) can be phosphorylated and phosphorylation generates high levels of actin-activated ATPase activity and actin motility. The other pair of myosin light chains consists of the two 17-kDa essential chains (LC17), the functional significance of which is unclear [1]. The regulatory and essential light chains are bound to an 8-nm stretch of myosin heavy chain, mostly a helix. This complex constitutes a regulatory domain [2,3] and is thought to act as a lever arm during force production by muscle motor proteins [4].The essential light chain, LC17, has two isoforms, the ratio of which depends on the tissue. The difference between these two species is five amino acid substitutions in the nine C-terminal residues (Fig. 1). In particular, one isoform, LC17a, has a charged glutamic acid residue at position 142, whereas the other, LC17b, has an alanine residue in the corresponding position [5]. An inverse correlation has been found between relative LC17b content and the maximal shortening velocity of skinned smooth muscle fibers [6] but this result is controversial [1]. Recently, weak but significant LC17 isoform-specific effects on the mechanical properties of smooth muscle cells [7] and strips [8] have been shown. In addition, the two isoforms have different subcellular distributions suggesting that they may have different functions [9].It has been shown that substitution mutations in the essential light chain of nonmuscle myosin from Dictyostelium may lead to decreases in actin-activated ATPase activity and motor function, as assessed by an in vitro motility assay [10].Katoh and co-workers [11,12] recently reported an efficient method for essential light chain exchange in smooth muscle myosin. We used this method to exchange LC17a, LC17b and mutated essential light chains with endogenous light chains from rabbit small-intestine myosin. The recombinant proteins were prepared ( Fig. 1; ELC1±6) with a histidine tag on their N-terminus to facilitate purification. In...

Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction

“…An inverse relationship between LC 17b content of SMs and both the V max of shortening of skinned SM fibers (39) and actin-activated myosin ATPase activity (22) has been reported. In contrast, exchanging the LC 17a isoforms on fully inserted SM-B myosin from intestine with the LC 17b isoform had no effect on the actin-activated ATP hydrolysis (50). This would suggest that the 7-amino acid insert is primarily responsible for the increased V max of actin-activated ATP hydrolysis (29), the physiological equivalent of the velocity of force generation.…”

Mechanisms of Vascular Smooth Muscle Contraction and the Basis for Pharmacologic Treatment of Smooth Muscle Disorders

“…Changes in the ELC17 could affect the stiffness of the SM MHC lever arm, and thus myosin step size and or unitary force. However for ELC17 isoforms, the motility assay does not show any difference in the velocity of actin translocation ( Kelley et al, 1993 ; Quevillon-Cheruel et al, 2000 ). Nonetheless in fast smooth muscle, the expression of ELC17a and SMA is higher than in slow smooth muscle ( Malmqvist and Arner, 1991 ).…”

Urinary Bladder Contraction and Relaxation: Physiology and Pathophysiology

“…Extraction/reconstitution of essential light chain isoforms in muscle fibers and overexpression in isolated cells have shown a slowing effect of LC 17b on contractile kinetics (288,457). Other comparative studies on isolated cells (586), in vitro motility experiments using expressed myosin heavy meromyosin (HMM) fragments, and essential light chain exchange experiments on isolated myosin have failed to show effects of the essential light chain composition on cell shortening velocity, ATPase, and actin translocation velocity (335,553). In view of the negative in vitro motility data regarding effects of essential light-chain exchange and the strong evidence for effects of myosin heavy chain insert, the essential light chain isoform expression does not seem to be the primary modulator of contractile kinetics in smooth muscle.…”