Abstract
We examined by a statistical approach the decrease of the Ca current (“run-down”) during long-lasting recordings with the whole-cell patch-clamp technique in guinea pig ventricular myocytes. The results are as follows. (1) Run-down of the Ca current (I Ca) occurs in three phases (T1–T3). T1 (38±19 min,n=135) and T3 (35±17 min,n=23) are characterized by a slow rate of decay ofI Ca [90±20 and 60±20 nA·cm−2·min−1, respectively]. T1 and T3 are separated by T2 (6±4 min,n=135) during which the current decays quickly [1200±230 nA·cm−2·min−1]. Between the onsets of T1 and T3,I Ca decreases from 11±3 to 3.5±1 μA/cm2. (2) Normalized current-voltage relationship, reversal potential and voltage-dependencies of steady-state activation and inactivation ofI Ca are globally shifted toward more negative potentials during the run-down process by 10–15 mV. (3)I Ca3 measured during T3 retains the pharmacological properties (blockade by D600, NiCl2 and CoCl3, increase by isoprenaline and insensitivity to tetrodotoxin) of the originalI Ca. (4) Intracellular perfusion of the nonhydrolysable ATP analogue AMP-PNP does not prevent the occurrence of T2, suggesting that a phosphorylation-dephosphorylation process is not involved in the fast run-down ofI Ca. (5) With 0.1 mM EGTA in the pipette, addition of 3 mM ATP significantly prolongsI Ca survival. No improvements are obtained by increasing the ATP concentration to 10 mM or replacing ATP with creatine phosphate. With 3 mM ATP present, increasing the EGTA concentration to 10–20 mM doublesI Ca survival time. EGTA alone (10 mM) is less effective than the mixture 3 mM ATP-0.1 mM·EGTA. Intracellular perfusion with a cytoplasmic extract considerably prolongs T2 and the overallI Ca survival. (6) The results are consistent with the hypothesis that run-down ofI Ca can partially be explained by a rise in intracellular Ca concentration and a loss of high energy compounds. Beneficial effect of ATP might include an increased capability of the cells to either extrude or sequester intracellular Ca, and a protection against enzymatic proteolysis.
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Recipient of successive fellowships from the Simone et Cino del Duca and Alexander von Humboldt Foundations
This work was supported by the Deutsche Forschungsgemeinschaft, SFB 246, Project A1
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Belles, B., Malécot, C.O., Hescheler, J. et al. “Run-down” of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium. Pflugers Arch. 411, 353–360 (1988). https://doi.org/10.1007/BF00587713
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DOI: https://doi.org/10.1007/BF00587713