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The vasodilator-stimulated phosphoprotein (VASP) functions as a cellular regulator of actin dynamics. VASP may initialise actin polymerisation, suggesting a direct interaction with monomeric actin. The present study demonstrates that VASP directly binds to actin monomers and that complex formation depends on a conserved four amino acid motif in the EVH2 domain. Point mutations within this motif drastically weaken VASP/G-actin interactions, thereby abolishing any actin-nucleating activity of VASP. Additionally, actin nucleation was found to depend on VASP oligomerisation since VASP monomers fail to induce the formation of actin filaments. Phosphorylation negatively affects VASP/G-actin interactions preventing VASP-induced actin filament formation.

Ca(2+) of 0.3-1.0 microM induces both the exposure of tryptic cleavage sites within the gelsolin molecule inaccessible in the Ca-free conformation, and binding of one actin monomer to the N-terminal half of gelsolin. On the other hand, gelsolin-induced enhancement of pyrene actin fluorescence was observed only above 50 microM Ca(2+), and a ternary actin/gelsolin complex preformed in 200 microM Ca(2+) was stable only above 30 microM Ca(2+). These results provide direct evidence for Ca-induced transitions from closed to open conformation of the gelsolin molecule in the range of 3 x 10(-7) to 10(-6) M Ca(2+). They also suggest that Ca(2+)>10(-5) M is required to stabilize actin-actin contacts in the 2:1 actin/gelsolin complex.

With the use of the patch-clamp technique, physiological mechanisms of Na+ channel regulation involving submembranous actin rearrangements were examined in human myeloid leukemia K562 cells. We found that the actin-severing protein gelsolin applied to cytoplasmic surface of membrane fragments at a high level of [Ca2+]i (1 microM) increased drastically the activity of Na-selective channels of 12 pS unitary conductance. In the experiments on intact cells, the elevation of [Ca2+]i using the ionophore 4Br-A23187 also resulted in Na+ channel activation. Addition of actin to the cytoplasmic surface of membrane patches reduced this activity to background level, likely due to actin polymerization. Our data imply that Ca-dependent modulations of the actin cytoskeleton may represent one of the general mechanisms of channel regulation and cell signalling.

A protein of Mr approximately 85 000 has been isolated and purified from pig stomach smooth muscle that modulates the polymer state of actin in a Ca2+-dependent manner. When added either to performed F-actin filaments or to G-actin, prior to polymerisation, the modulator induces the formation of shorter filaments. The average filament length in the presence of the modulator is directly dependent on its molar ratio to actin indicating a stoichiometric rather than a catalytic type of interaction. When mixed with G-actin the modulator forms a stable complex with two actin monomers; this complex is presumed to act as a potent nucleus for actin polymerisation. The dynamics of the interaction with F-actin suggests a direct severing of actin filaments by the modulator via a binding to intrafilamentous actins.

The actin-modulating protein from pig stomach smooth muscle (PSAM) which reduces the average filament length has two opposite effects on the interaction of actin with skeletal muscle myosin: (1) stimulation of both the Mg2+-ATPase activity and superprecipitation at low KCl concentrations, and (2) inhibition of these two interrelated processes at an ionic strength close to physiological. Both stimulation and inhibition were Ca2+-dependent, reflecting the requirement for Ca2+ for the interaction of the modulator with actin. With acto-subfragment-1, only inhibition of the actin-activated ATPase was observed. Possible implications of these effects for studies on the regulation of smooth muscle contraction are discussed.

Gelsolin is a calcium-dependent actin severing and capping protein. Calcium 'opens' the molecule to make actin binding sites accessible, but removal of calcium from the medium does not necessarily fully reverse this process. The calcium sensitivity of actin monomer binding and actin filament severing is here shown to vary considerably with the source of gelsolin and conditions of preparation. Plasma gelsolin undergoes irreversible loss of calcium sensitivity when prepared in the presence of calcium ions. This is not due solely to effects of bound calcium, because purified human plasma gelsolin expressed in E. coli and stored in calcium shows no comparable loss of calcium sensitivity when prepared or stored in calcium. These results suggest the presence of factors in plasma which, in the presence of calcium, promote an irreversible structural change in gelsolin resulting in permanent loss of calcium sensitivity.