THE EFFECT OF IONIC-STRENGTH ON THE KINETICS OF RIGOR DEVELOPMENT IN SKINNED FAST-TWITCH SKELETAL-MUSCLE FIBERS

Recent atomic 3-D reconstructions of the acto-myosin interface suggest that electrostatic interactions are important in the initial phase of cross-bridge formation. Earlier biochemical studies had also given st rong evidence for the ionic strength dependence of this step in the cr oss-bridge cycle. We have probed these interactions by altering the io nic strength (Gamma/2) of the medium mainly with K+, imidazole(+) and EGTA(2-) to vary charge shielding. We examined the effect of ionic str ength on the kinetics of rigor development at low Ca2+ (experimental t emperature 18-22 degrees C) in chemically skinned single fast-twitch f ibres of mouse extensor digitorum longus (EDL) muscle. On average the delay before rigor onset was 10 times longer, the maximum rate of rigo r tension development was 10 times slower, the steady-state rigor tens ion was 3 times lower and the in-phase stiffness was 2 times lower at high (230 mM) compared to low (60 mM) ionic strength. These results we re modelled by calculating ATP depletion in the fibre due to diffusion al loss of ATP and acto-myosin Mg.ATPase activity. The difference in d elay before rigor onset at low and high ionic strength could be explai ned in our model by assuming a 15 times higher Mg.ATPase activity and a threefold increase in K-m in relaxing conditions at low ionic streng th. Activation by Ca2+ induced at different time points before and dur ing onset of rigor confirmed the calculated time course of ATP depleti on. We have also investigated ionic strength effects on rigor developm ent with the activated troponin/tropomyosin complex. ATP withdrawl at maximum activation by Ca2+ induced force transients which led into a ' 'high rigor'' state. The peak forces of these force transients were ve ry similar at low and high ionic strength. The subsequent decrease in tension was only 10% slower and steady-state ''high rigor'' tension wa s reduced by only 27% at high compared to low ionic strength. Addition of 10 mM phosphate to lower cross-bridge attachment strongly suppress ed the transient increases in force at high ionic strength and reduced the steady-state rigor tension by 17%. A qualitatively similar but sm aller effect of phosphate was observed at low ionic strength where ste ady-stale rigor force was reduced by 10%. The data presented in this s tudy show a very strong effect of ionic strength on rigor development in relaxed fibres whereas the ionic strength dependence of rigor devel opment after thin filament activation was much less. The data confirm the importance of electrostatic interactions in cross-bridge attachmen t and cross-bridge-attachment-induced activation of thin filaments.