Myosin Essential Light Chain Isoforms Modulate the Velocity of Shortening Propelled by Nonphosphorylated Cross-bridges

Abstract

The differential effects of essential light chain isoforms (LC 17a and LC 17b ) on the mechanical properties of smooth muscle were determined by exchanging recombinant for endogenous LC 17 in permeabilized smooth muscle treated with trifluoperazine (TFP). Co-precipitation with endogenous myosin heavy chain verified that 40 -60% of endogenous LC 17a could be exchanged for recombinant LC 17a or LC 17b . Upon addition of MgATP in Ca 2؉ -free solution, recombinant LC 17 exchange induced slow contractions unaccompanied by regulatory light chain (RLC) phosphorylation only in TFP-treated, but not in untreated, permeabilized smooth muscle; the shortening velocity and rate of force development were approximately 1.5 and 2 times faster, respectively, in response to LC 17a than LC 17b . Additional incubation with recombinant, thiophosphorylated RLC increased the shortening velocity, independent of the LC 17 isoform exchanged. The LC 17 -induced contractions of TFPtreated muscles were abolished by prior addition of nonphosphorylated RLC. We suggest that LC 17 stiffens the lever arm of myosin and, in the absence of regulation by RLC, permits cross-bridge cycling without requiring RLC phosphorylation. Our results are compatible with nonphosphorylated RLC acting as a repressor and with LC 17 isoforms modulating the MgADP affinity and, consequently, rate of cooperative cycling of nonphosphorylated cross-bridges.The markedly different rates of contraction and relaxation in fast, phasic and slow, tonic smooth muscles reflect differences not only at the level of the membrane potential, signal transduction, rates of myosin light chain phosphorylation and dephosphorylation, but also in the kinetic properties of the actomyosin motor. The muscle-specific differences in actomyosin kinetics have been ascribed to the existence of specific myosin isoforms, but their relative contributions to the mechanical properties of smooth muscles have not been unequivocally demonstrated (reviewed in Ref. 1). The expression of the two myosin heavy chain (MHC) 1 isoforms, SM-1 and SM-2 (2) that differ by, respectively, the presence or absence of a 34-amino acid extension of the carboxyl terminus (3), does not correlate with phasic or tonic kinetics of contraction. On the other hand, motility assays show different rates of movement propelled by MHC isoforms that are the products of an alternative splicing mechanism resulting in the presence or absence of a 7-amino acid insert near the nucleotide-binding region of the myosin head (4, 5). Disparate results have been obtained concerning the effects on contractile kinetics of the two LC 17 isoforms, acidic (LC 17a ) and basic (LC 17b ), that differ in 5 of the 9 COOHterminal amino acid residues and are products of a single gene generated by an alternative splicing mechanism (6). The faster kinetics of phasic, smooth muscle in situ (7-9) correlates with the expression of the LC 17a isoform (10, 11), albeit the proportion of myosin heavy chain containing the insert was also variant in these muscles, whereas ...

Cystic fibrosis transmembrane conductance regulator (CFTR) dependent cytoskeletal tension during lung organogenesis

“…In the CFTR-treated fetal lungs, however, the basic form is the predominant form. The acidic form of LC 17 is associated with phasic contractions of immature smooth muscle while the basic form is characteristic of tonic contractions of mature smooth muscle (Malmqvist and Arner, 1991;Matthew et al, 1998). Thus, CFTR overexpression alters expression of the essential LC 17 isoforms that are associated with a mature smooth muscle phenotype and increases the LC 20 phosphorylation required for contraction.…”

Forced Expression of Essential Myosin Light Chain Isoforms Demonstrates Their Role in Smooth Muscle Force Production

“…The molecular basis for tonic and phasic contractile properties is unknown, but has been suggested to be regulated by a variety of factors including splice variant isoforms of the myosin heavy chain (MHC) 1 at the 25/50-kDa junction (1) and/or splice variant isoforms of the essential myosin light chain (2) producing an acidic (MLC 17a ) and basic (MLC 17b ) isoform. The rate of force activation (6,7), V max (6,7), the velocity of actin movement in the in vitro motility assay (1,8), and ATPase activity (1,9) have been correlated with the expression of splice variant isoforms both of the MHC and MLC 17 . In this study, we tested the hypothesis that the mechanical properties of smooth muscle are regulated by splice variant expression of MLC 17 .…”

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

“…Little [41] or no [35] difference has been reported in the actin-activated ATPase activity of myosins enriched in LC17a or LC17b at high actin concentration. A small increase (by a factor of 1.5±2) in response to LC17a, but not LC17b, has also been reported for some parameters of the contractile properties of smooth muscle cells or strips [7,8].…”

“…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].…”