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Abstract

Using the intensity of the outer part of the second actin layer line as an indicator of thin filament conformation in vertebrate muscle we were able to identify the four different states of rest, and the three states induced by the presence of Ca2+ ions, rigor bridge attachment and actively cycling bridges, respectively. These findings are in qualitative agreement with a number of biochemical studies by Eisenberg and Greene and others, indicating that activation of the thin filament depends both on Ca2+ ions and crossbridge binding. Yet quantitatively, the biochemical data and our structural data are contradictory. Whereas the biochemical studies suggest a strong coupling between structural changes of the thin filament and the ATPase activity, the structural studies indicate that this is not necessarily the case. Troponin molecules also change their conformation upon activation depending on both Ca2+ ions and crossbridge binding as demonstrated by the early part of the time course of the thin filament meridional reflections in contracting frog muscle. Low ionic strength which has been shown by Brenner and collaborators to increase weakly binding crossbridges in relaxed rabbit psoas muscle does not influence the intensity of the second actin layer line in this muscle. Yet in contracting frog muscle the increase of the second actin layer line increases very rapidly in one step, suggesting that weak binding bridges which are attached to actin prior to force production may indeed influence the thin filament conformation. It therefore appears that weakly bound bridges in the low ionic strength state do not have the same effect on the thin filament conformation as weakly bound bridges in an actively contracting muscle. Arthropod muscles like the thin filament regulated lobster muscles differ from vertebrate muscle in not showing an increase of the second layer line during contraction, which may have to do with differences in crossbridge attachment. The myosin-regulated molluscan muscle ABRM shows a large increase on the second actin layer line upon phasic contraction and a much smaller increase in catch or rigor, indicating that actively cycling bridges influence the thin filament conformation differently than catch or rigor bridges. Several pieces of evidence which we have briefly outlined in this paper suggest that the thin filament conformational changes we have observed do not arise solely from tropomyosin movements and that conformational changes of actin domains should be considered.