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

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 (ICa) 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 of ICa [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, ICa decreases from 11 +/- 3 to 3.5 +/- 1 microA/cm2. (2) Normalized current-voltage relationship, reversal potential and voltage-dependencies of steady-state activation and inactivation of ICa are globally shifted toward more negative potentials during the run-down process by 10-15 mV. (3) ICa3 measured during T3 retains the pharmacological properties (blockade by D600, NiCl2 and CoCl3, increase by isoprenaline and insensitivity to tetrodotoxin) of the original ICa. (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 of ICa. (5) With 0.1 mM EGTA in the pipette, addition of 3 mM ATP significantly prolongs ICa survival. No improvements are obtained by increasing the ATP concentration to 10 mM or replacing ATP with creatine phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)