CHARACTERIZATION AND PARTIAL-PURIFICATION OF THE CYTOPLASMIC FACTOR THAT MAINTAINS CARDIAC CA2+ CHANNEL ACTIVITY

Using the patch clamp method we attempted to characterize the cytoplas mic factor in guinea-pig cardiac myocytes which restores L-type Ca2+ c hannel activity after run-down. The factor was eluted from a diethylam inoethyl (DEAE) sepharose column bq KCl at 100-360 mM. On gel filtrati on the factor had an apparent molecular mass (M-r) of 250-300 kDa, Two -dimensional electrophoresis of the partially purified factor showed a t least nine spots, of which the major spot had a M-r of about 100 kDa and an isoelectric point of 4.8, suggesting that the physicochemical properties of the factor resemble those of calpastatin, an endogenous inhibitor of Ca2+-activated protease, calpain. Calpastatin activity wa s increased in the partially purified cytoplasm and an antibody raised against calpastatin recognized the major band. Reduction of calpastat in in the cytoplasm decreased the potency of Ca2+ channel activation. These results suggest that calpastatin might interact with the Ca2+ ch annel and maintain channel activity.