Cation effects on the conformations of muscle and non-muscle α-actinins

Abstract

We examined the effects of changing KCl concentration on the secondary structures of α-actinins using circular dichroism (CD), 1,1′-bis(4-anilino) naphthalene-5,5′-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mm KH₂PO₄, pH 7.5, increasing KCl from 0 to 120 mm led to decreases in α-helix conformation for brain, platelet and heart α-actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mm KCl, 0.65 mm calcium produced small changes in the CD spectra of both brain and platelet α-actinin, but had no effect on heart α-actinin. bisANS fluorescence of all three α-actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mm KCl increasing concentrations of Ca²⁺ or Mg²⁺ did not have significant effects on the bisANS fluorescence of any α-actinin. Digestion of brain, platelet and heart α-actinins with α-chymotrypsin showed an increase of proteolytic susceptibility in 120 mm KCl. These experiments also showed that increasing the concentration of Ca²⁺ or Mg²⁺ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mm KCl. The results suggest that α-actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different α-actinins.