Characterization and partial purification of the cytoplasmic factor that maintains cardiac Ca2+ channel activity.
Using the patch clamp method we attempted to characterize the cytoplasmic factor in guinea-pig cardiac myocytes which restores L-type Ca2+ channel activity after run-down. The factor was eluted from a diethylaminoethyl (DEAE) sepharose column by KCl at 100-360 mM. On gel filtration the factor had an apparent molecular mass (Mr) of 250-300 kDa. Two-dimensional electrophoresis of the partially purified factor showed at least nine spots, of which the major spot had a Mr 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 was increased in the partially purified cytoplasm and an antibody raised against calpastatin recognized the major band. Reduction of calpastatin in the cytoplasm decreased the potency of Ca2+ channel activation. These results suggest that calpastatin might interact with the Ca2+ channel and maintain channel activity.
Kameyama
A
A
Department of Physiology, Faculty of Medicine, Kagoshima University, Kagoshima 890, Japan.
Hao
L Y
LY
Takano
E
E
Kameyama
M
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D002417:Cattle; D002458:Cell Fractionation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000648:ultrastructure; D008970:Molecular Weight; D009206:Myocardium / Q000648:ultrastructure*; D018408:Patch-Clamp Techniques; D011189:Potassium Chloride
D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D011189:Potassium Chloride; C033668:calpastatin; D000255:Adenosine Triphosphate