Blebbistatin Effects Expose Hidden Secrets in the Force-Generating Cycle of Actin and Myosin

Mohammad A Rahman,Marko Ušaj,Dilson E Rassier,Alf Månsson

doi:10.1016/j.bpj.2018.05.037

Mohammad A Rahman, Marko Ušaj + Show 2 more

Open Access

https://doi.org/10.1016/j.bpj.2018.05.037

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Journal: Biophysical Journal	Publication Date: Jul 1, 2018
Citations: 36	License type: cc-by

Affiliation: Linnaeus University, McGill University

Abstract

Cyclic interactions between myosin II motors and actin filaments driven by ATP turnover underlie muscle contraction and have key roles in the motility of nonmuscle cells. A remaining enigma in the understanding of this interaction is the relationship between the force-generating structural change and the release of the ATP-hydrolysis product, inorganic phosphate (Pi), from the active site of myosin. Here, we use the small molecular compound blebbistatin to probe otherwise hidden states and transitions in this process. Different hypotheses for the Pi release mechanism are tested by interpreting experimental results from in vitro motility assays and isolated muscle fibers in terms of mechanokinetic actomyosin models. The data fit with ideas that actomyosin force generation is preceded by Pi release, which in turn is preceded by two serial transitions after/coincident with cross-bridge attachment. Blebbistatin changes the rate limitation of the cycle from the first to the second of these transitions, uncovering functional roles of an otherwise short-lived pre-power stroke state that has been implicated by structural data.

Highlights

Muscle contraction results from cyclic interactions of myosin II motor domains with actin filaments driven by ATP turnover
The half-maximal inhibition occurred in the range of $1–5 mM blebbistatin for three different batches of heavy meromyosin (HMM) and three different batches of blebbistatin with a mixture of (þ) and (À) enantiomers
We titrated the concentration of each specific blebbistatin batch to achieve $50% blebbistatin-induced reduction in velocity compared to the control

Summary

Introduction

Muscle contraction results from cyclic interactions of myosin II motor domains with actin filaments driven by ATP turnover. This process underlies bodily movements powered by skeletal muscle, pumping of blood by the heart, and a range of homeostatic mechanisms governed by smooth muscle. Disturbed myosin II function is central to a range of diseases [2,3,4] as well as to functional decline during aging [5]. The insights into diseases as well as the associated drug discovery processes are hampered by remaining enigmas in the fundamental understanding of the force-generating cross-bridge cycle between myosin II and actin. Outstanding questions include but are not limited to the following. 1)

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