C. Veigel et al., THE EFFECT OF IONIC-STRENGTH ON THE KINETICS OF RIGOR DEVELOPMENT IN SKINNED FAST-TWITCH SKELETAL-MUSCLE FIBERS, Pflugers Archiv, 435(6), 1998, pp. 753-761
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.