calpain and 1-4-7-10-tetraazacyclododecane--1-4-7-10-tetraacetic-acid

We report the development of a novel procedure for generating fast, high-frequency Ca2+ oscillations in vitro and the frequency-dependent activation of m-calpain, the Ca2+-activated intracellular cysteine protease. The procedure is based upon liberating Ca2+ from a cage, DM-Nitrophen, by repetitive UV laser pulses and its concomitant binding by a 'slow' chelator, 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA). It is shown that a full control over the pattern of oscillations can be readily achieved because the half-life of individual spikes is determined by DOTA concentration and pH, whereas peak amplitude can be adjusted by light intensity. Frequency is only limited by the physical parameters of the light source. The sensitivity of calpain activation to the frequency of Ca2+ oscillations was monitored by the cleavage of microtubule-associated protein 2, a very sensitive physiological substrate of the enzyme. One hundred transients at a peak Ca2+ concentration of 10 microM were presented at various pH values and frequencies ranging from 1 to 50 Hz. At pH 6.0 and 7.0 significant activation occurred at high frequencies (20 and 50 Hz), but here Ca2+ accumulated due to the overlap of transients; at low frequencies (1 and 3 Hz) where Ca2+ accumulation was negligible, there was no calpain activation. At pH 8.0, where individual transients do not overlap even at 50 Hz, frequency-dependence of activation is seen when calpain is sensitized to Ca2+ by autolysis and by the addition of a phospholipid, phosphatidylinositol-4,5-bisphosphate. Our results show that calpain is sensitive to the frequency of fast Ca2+ oscillations in vitro, which is of potential physiological significance.

Topics: Calcium; Calpain; Chelating Agents; Heterocyclic Compounds, 1-Ring; Humans; Neurons; Signal Transduction