Vanadate Responses of Insect Fight Muscle

Abstract

When exposed to sodium orthovanadate (Vi), permeabilized insect flight muscle (IFM) from Lethocerus water bugs behaves differently in three ways from vertebrate skeletal muscle (VSkM) under similar conditions. Weak binding of Vi in RLX: IFM treated 5-10 min with 250 µM Vi in Lo-µ RLX buffer (= Vi-RLX) (pCa ∼9.0; µ ∼90 mM), then washed free of Vi in Lo-µ RLX, and placed in Lo-µ ACT buffer (pCa ∼4.5; µ ∼105 mM) rises to maximal isometric tension far more slowly (fives of minutes) than untreated IFM, suggesting a weak-binding, slowly reversible interaction with (presumably) myosin. IFM 100x more sensitive to Vi than VSkM: Exposing maximally Ca-activated IFM to 250 mM Vi in Lo-µ ACT (= Vi-ACT) quickly suppresses active-state tension 97-100% and induces relaxed-state X-ray diffraction structure. Vi-ACT-exposed IFM recovers little or no active-state tension during 30-60 subsequent minutes in Lo-µ ACT. Vi-trapping in IFM is so strong that just 2 µM Vi ultimately (30-50 min) suppresses active tension in Lo-µ ACT by ∼75%, suggesting that 50% IFM force inhibition would require 1 µM or less Vi, versus 45-94 µM in VSkM. Only crossbridges opposite target zones are Vi-trapped: Despite 97-100% paralysis of Ca-activated isometric force production by Vi-ACT, Vi trapping only affects myosin crossbridges opposite actin target zones. When stretched 2-4%, fibers recover active tension capability. Post-Vi washout with RLX, followed by 2-4% stretch, followed by Lo-µ ACT exposure, generates significant Ca-activated tension, because at the longer sarcomere length target zones have moved toward myosin heads that at rest length were unable to reach the 2/7 fraction of IFM actin monomers (Wu etal, PLoS One 5: e12643 (2010)) that accept strong-binding crossbridges, ATPase cycling and tight Vi trapping. Slow 3% length-cycling in Vi-ACT Vi-traps all accessible IFM crossbridges. (Support: NIH, MDA).