Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after “run-down”.The factors include ATP,calpastatin and H fraction(a high molecular fraction of bovine cardiac cytoplasm).Methods Single Ca2+ channel activities were recorded with patch clamp technique in guinea-pig cardiac myocytes.Run-down was induced by the inside-out patch formation.Calpastatin(CS),calmodulin(CaM)and three GST-fusion fragment peptides derived from the C-terminal tail of guinea-pig Cav1.2,CT-1(amino acids number 1509-1791),CT-2(1777-2003)and CT-3(1944-2169)were produced as GST fusion proteins.Results(1)CaM + ATP or CS + ATP restored the channels after run-down;however,the CaM or CS’s effects became smaller with the longer run-down time.(2)After run down,CaM-dependent protein kinase(CaMKII)produced Ca2+ channel activity to only 2-10% of the basal activity,however,in the presence of CaMKII,the time-dependent nature of the CaM effect was abolished.(3)In pull-down assay,CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with phosphatase.(4)CaMKII was detected in the H fraction of bovine cardiac cytoplasm.Conclusions The results show that CS,CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca2+ channels,and that there might be cross talks among the four factors(CS,CaM,CaMKII and the undefined cytoplasmic factor). Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2 + channel activity after “run-down”. The factors include ATP, calpastatin and H fraction (a high molecular fraction of bovine cardiac cytoplasm). Methods Single Ca2 + channel activities were recorded with patch clamp technique in guinea-pig cardiac myocytes. Run-down was induced by the inside-out patch formation. Calpastatin (CS), calmodulin (CaM) and three GST-fusion fragment peptides derived from the C-terminal tail of guinea-pig Cav1.2, CT-1 (amino acids number 1509-1791), CT-2 (1777-2003) and CT-3 (1944-2169) were produced as GST fusion proteins. Results or CS + ATP restored the channels after run-down; however, the CaM or CS’s effects became smaller with the longer run-down time. (2) After run down, CaM-dependent protein kinase (CaMKII) produced Ca2 + channel activity to only 2-10% of the basal activity, however, in the presence of CaMKII, the time-dependent nature of the CaM effect was abolished. (3) In pull-down a (4) CaMKII was detected in the H fraction of bovine cardiac cytoplasm. Conclusions The results show that CS, CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca2 + channels, and that there might might cross talks among the four factors (CS, CaM, CaMKII and the undefined cytoplasmic factor).