Force-generating cross-bridges during ramp-shaped releases: evidence for a new structural state.

Mechanical and two-dimensional (2D) x-ray diffraction studies suggest that during isometric steady-state contraction, strongly bound cross-bridges mostly occupy early states in the power stroke, whereas rigor or rigor-like cross-bridges could not be detected. However, it remained unclear whether cross-bridges accumulate, at least transiently, in rigor or rigor-like states in response to rapid-length releases. We addressed this question using time-resolved recording of 2D x-ray diffraction patterns of permeabilized fibers from rabbit psoas muscles during isometric contraction and when small, ramp-shaped length-releases were applied to these fibers. This maneuver allows a transient accumulation of cross-bridges in states near the end of their power stroke. By lowering the temperature to 5 degrees C, force transients were slowed sufficiently to record diffraction patterns in several 2-4-ms time frames before and during such releases, using the RAPID detector (Refined ADC Per Input Detector) at beam line ID02 of the European Synchrotron Radiation Facility (Grenoble, France). The same sequence of frames was recorded in relaxation and rigor. Comparisons of 2D patterns recorded during isometric contraction, with patterns recorded at different [MgATPgammaS] and at 1 degrees C, showed that changes in intensity profiles along the first and sixth actin layer lines (ALL1 and ALL6, respectively) allowed for discernment of the formation of rigor or rigor-like cross-bridges. During ramp-shaped releases of activated fibers, intensity profiles along ALL1 and ALL6 did not reveal evidence for the accumulation of rigor-like cross-bridges. Instead, changes in the ALL6-profile suggest that during ramp-shaped releases, cross-bridges transiently accumulate in a structural state that, to our knowledge, was not previously seen, but that could well be a strongly bound state with the light-chain binding domain in a conformation between a near prepower-stroke (isometric) orientation and the orientation in rigor.

Comparison of the effects of Ca2+ and those of Mg2+ and La3+ on endplate currents in mouse interosseus muscles.

Acetylcholine-induced currents were measured in dissociated muscle fibres from the mouse interosseus. Fast and slow desensitization could be distinguished, the latter ending in a steady-state current. The time constants of the two phases of current decrease and the steady-state current were decreased when the superfusing solution contained di- or trivalent cations in addition to the 0.2 mM Mg2+ present in the control solution. When Ca2+, Mg2+ or La3+ was added at the time at which the current had settled to a near steady-state, there was a fall of current, the time course of which was much faster than that during desensitization. The amplitude and the time course of current change were nearly the same for Ca2+ and Mg2+, showing that the effect was not specific for any particular cation. No evidence of intracellular action of the cations was found. The [ACh]-current relationship was shifted to a higher [ACh] by raising [Mg2+] or [La3+]. Recovery of the ability to react to ACh was slowed by increasing [La3+], but the initial recovery of fast and slow time constants of desensitization was not much changed. Recovery was impeded by the presence of a low (conditioning) [ACh]; this effect was enhanced by La3+. It was concluded that the effects of polyvalent cations are similar to those of non-specific blockers.