The 80 kDa human erythrocyte calpain, when exposed to Ca²⁺, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca²⁺ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E‐64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca²⁺, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to μM Ca²⁺, membrane‐bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca²⁺‐induced translocation to the inner surface of plasma membranes. In the membrane‐bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations.

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca²⁺-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca²⁺-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca²⁺-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca²⁺-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.

Persistent dysregulation in Ca²⁺ homeostasis is a pervasive pathogenic mechanism in most neurodegenerative diseases, and accordingly, calpain activation has been implicated in neuronal cells dysfunction and death. In this study we examined the intracellular functional state of the calpain-calpastatin system in −G93A(+) SOD1 transgenic mice to establish if and how uncontrolled activation of calpain can be prevented in vivo during the course of prolonged [Ca²⁺]_i elevation. The presented data indicate that 1) calpain activation is more extensive in motor cortex, in lumbar, and sacral spinal cord segments compared with the lower or almost undetectable activation of the protease in other brain areas, 2) direct measurements of the variations of Ca²⁺ levels established that the degree of the protease activation is correlated to the extent of elevation of [Ca²⁺]_i, 3) intracellular activation of calpain is always associated with diffusion of calpastatin from perinuclear aggregated forms into the cytosol and the formation of a calpain-calpastatin complex, and 4) a conservative fragmentation of calpastatin is accompanied by its increased expression and inhibitory capacity in conditions of prolonged increase in [Ca²⁺]_i. Thus, calpastatin diffusion and formation of the calpain-calpastatin complex together with an increased synthesis of the inhibitor protein represent a cellular defense response to conditions of prolonged dysregulation in intracellular Ca²⁺ homeostasis. Altogether these findings provide a new understanding of the in vivo molecular mechanisms governing calpain activation that can be extended to many neurodegenerative diseases, potentially useful for the development of new therapeutic approaches.

In resting human neuronal cells, nitric oxide synthase (nNOS) is present in its native 160 kDa form in a quiescent state predominantly co‐localized on the plasma membrane, via its PDZ (Psd‐95/Discs‐large/Zona Occludens) domain, with NMDA receptor (NMDA‐R) and in tight association with heat shock protein 90 (HSP90). Following exposure of the cells to Ca²⁺‐ionophore or to NMDA, nNOS undergoes proteolytic removal of the PDZ domain being converted into a fully active 130 kDa form. The newly generated nNO synthase form dissociates from NMDA‐R and extensively diffuses into the cytosol in direct correlation with NO production. Intracellular redistribution and activation of nNOS are completely prevented in cells preloaded with calpain inhibitor‐1, indicating that these processes are triggered by a concomitant activation of calpain. The role of calpain has been confirmed by immunoprecipitation experiments revealing that also μ‐calpain is specifically recruited into the NMDA‐R‐nNOS‐HSP90 complex following calcium loading. Thus, the formation of clusters containing HSP90, μ‐calpain, nNOS and NMDA‐R represents the limiting step for the operation of the mechanism that links an efficient synthesis of NO to a local increase in Ca²⁺ influx.