Podocytes are epithelial cells that line the outer aspect of renal blood vessels and provide a platform for the kidney's filtering apparatus, the slit diaphragm. Mutations in alpha-actinin-4, an actin bundling protein highly expressed in podocytes, result in increased affinity for actin and cause a familial form of focal segmental glomerulosclerosis. We hypothesized that such gain-of-affinity mutations would override alpha-actinin-4's sensitivity to regulatory factors such as calcium (acting via two EF-hand motifs), and phosphoinositides. We generated calcium- (mutEF) and phosphoinositide- (mutPI) insensitive variants of alpha-actinin-4, comparing their properties to a disease-associated mutant (K256E) and to the wildtype (wt) protein. alpha-Actinin-4(mutPI) displayed increased affinity for actin, while the affinity of alpha-actinin-4(mutEF) was unchanged. Addition of calcium to actin sedimentation assays caused a decrease in the association of alpha-actinin-4(wt) with filamentous actin, while phosphoinositides generally increased this association. Similar to alpha-actinin-4(K256E), alpha-actinin-4(mutPI) was mislocalized in cultured podocytes, being preferentially associated with filamentous actin and focal adhesions. Fluorescence recovery after photobleaching experiments revealed a rapid turnover of alpha-actinin-4(wt) and alpha-actinin-4(mutEF) along stress fibers and focal adhesions, while the turnover of alpha-actinin-4(K256E) and alpha-actinin-4(mutPI) was dramatically reduced at these subcellular locales. Equibiaxial mechanical stimulation of podocytes, a mimic of intraglomerular forces, reduced podocyte surface area by 50%; this decrease was more severe (70%) in the presence of high-affinity mutants of alpha-actinin-4. These data suggest that dynamic regulation of alpha-actinin-4/actin interactions may be necessary for maintaining podocyte structure in response to glomerular hydrostatic forces.