Run-down of the cardiac Ca 2+ channel: characterization and restoration of channel activity by cytoplasmic factors

Abstract

Possible mechanisms for run-down in the Ca2+ channel, such as proteolysis or dephosphorylation of the channel, were examined in guinea-pig ventricular myocytes. The Ca2+ channel current, recorded in inside-out patches using a pipette solution containing 50 mM Ba2+ and 3 microM Bay K 8644, ran down with a mean survival time of 2.35 min. The survival time was not significantly affected by adenosine triphosphate (ATP) (3 mM), 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA) (2 mM), isoprenaline (l-5 microM), phosphate (l20 mM) and leupeptin (l0 microM). Stimulation of guanosine triphosphate (GTP)-binding proteins was also ineffective. The catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA, 0.5-2 microM) slightly and transiently increased channel activity, but had minimal effects on the channel when applied after complete run-down. On the other hand, cytoplasm from the heart, skeletal muscle, brain and liver, but not kidney, induced channel activity. There was a positive correlation between NPo (the product of the number of channels N and the open probability Po) value before run-down and that after the application of cytoplasm, suggesting that the activity of once-active channels was restored ba the exogenous cytoplasm. The potency of cytoplasm in tissues in inducing channel activity was not related to PKA activity nor to the number of dihydropyridine binding sites. These results suggest that the run-down of the cardiac Ca2+ channel is not mediated by dephosphorylation or proteolysis of the channel, but involves other factor(s), possibly interaction of the channel protein with a cytoplasmic regulatory protein.

Cytochalasin D reduces Ca2+ currents via cofilin‐activated depolymerization of F‐actin in guinea‐pig cardiomyocytes

“…In control conditions (i.e. in the absence of pharmacological interventions) peak I Ca was not stable but slowly ran down (Table 1, Control), in accordance with the literature (Kameyama et al 1997).…”

Calmodulin Kinase II Activation Is Required for the Maintenance of Basal Activity of L-Type Ca2+ Channels in Guinea-Pig Ventricular Myocytes

“…The stock solution of protease inhibitors (×1000 concentration, protease inhibitor cocktail) contained 10 mM benzamidine, 10 mg / ml iodoacetamide, 10 mM PMSF, 2 mg / ml aprotinin, 5 mg / ml leupeptin, and 0.5 mg / ml pepstatin A (3,8). The homogenate was centrifuged at 10,000 × g for 20 min and then at 100,000 × g for 30 min.…”

“…Recent work also shows that phosphatidylinositol-4,5-bisphosphate (PIP 2 ) can stabilize the activity of P / Q-type Ca 2+ channels (5,6). Alternatively, we have reported that a cytoplasmic extract from cardiac tissue recovers the Ca 2+ channel activity from run-down (3,7,8), suggesting that run-down of the Ca 2+ channel activity may be due to the washout of certain cytoplasmic factors. Subsequently the cytoplasmic factors were identified as calpastatin, ATP, and a yet unidentified factor in a high molecular mass fraction (H fraction) obtained from gel filtration of the cardiac tissue (3,9,10).…”

Complete reversal of run-down in rabbit cardiac Ca 2+ channels by patch-cramming in Xenopus oocytes; partial reversal by protein kinase A

“…Inclusion of ATP, the catalytic subunit of protein kinase A (PKAc), or other agents that promote phosphorylation has been shown to dramatically decrease, and in some cases reverse, the run-down of whole-cell currents [6,8,9,10]. At the single-channel level, PKAc has been reported to prevent the run-down of HVA channels in the GH 3 cell line [11] and in myocytes [12], although there is conflicting evidence that PKAc prolongs activity in myocytes longer than in control excisions [13]. Yatani et al [14] found that a mixture of compounds that activate G-proteins and promote phosphorylation is able to reverse the run-down of cardiac HVA single channels in excised patches.…”