?Run-down? of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium

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.