Metabolic inhibition reduces cardiac L-type Ca2+ channel current due to acidification caused by ATP hydrolysis

Figures

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  Fig 1. Effect of FCCP on isoprenaline stimulated ICa,L. (A) Effect of FCCP on isoprenaline (ISO, 1 μM)-stimulated ICa,L. Traces of ICa,L shown on the top were recorded at the times indicated by the corresponding letters on the main graph. (B) Concentration-response curve for the effect of FCCP on ISO-stimulated ICa,L. (C) Current-voltage (I-V) relationships (a) and inactivation curves (b) of ICa,L under basal conditions, during ISO (1 μM) stimulation and during exposure to two different FCCP concentrations (70 and 100 nM) in the presence of ISO. Inset: Double-pulse protocol used for the inactivation curves (see Methods for details). ICa,L peak amplitude during S2 pulse is expressed as a percentage of ICa,L measured at 0 mV without conditioning pulse S1 and plotted as a function of S1 pulse potential.

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  Fig 2. Effect of various metabolic inhibitors on isoprenaline stimulated ICa,L. Peak amplitude of ISO-stimulated ICa,L during exposure of the cells to various inhibitors of oxidative phosphorylation. Values are presented as means ± SEM for the number of cells indicated in parentheses. * P<0.05 vs. ISO alone.

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  Fig 3. Effect of intracellular ATP on ICa,L amplitude. (A) A typical experiment representing the effect of ISO (1 μM) on basal ICa,L in the absence of ATP in pipette solution. The current traces shown on the top of the panel were recorded at the times indicated by the corresponding letters on the main graph. (B) Mean basal and ISO-stimulated ICa,L amplitudes recorded in cells dialyzed with 3 mM ATP and ATP-free internal solutions. *P<0.05, **P<0.01 for the number of cells indicated in parentheses.

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  Fig 4. Effect of oligomycin on ICa,L and its response to FCCP. (A) A typical experiment showing the effect of a pretreatment of the cell with oligomycin (30 μM) on ISO (1 μM)-stimulated ICa,L and on the response to FCCP. (B) Mean amplitude of ISO-stimulated ICa,L recorded in the absence and presence of FCCP (100 nM) and oligomycin (30 μM). *P<0.05, **P<0.005.

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  Fig 5. Role of channel phosphorylation in the effect of FCCP on ICa,L. (A) Comparison of the effect of a metabolic inhibition induced by FCCP on ICa,L stimulated by either ISO (1 μM) or intracellular cAMP (20 μM). (B) Comparison of the effect of FCCP on ISO-stimulated ICa,L in the presence or absence of ATP-γ-S (3 mM) in the pipette-filling solution. * P<0.05 and # p<0.05 vs absence of FCCP.

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  Fig 6. Non-hydrolysable ATP analogues prevent decrease of ICa,L by metabolic inhibition. (A) A typical experiment showing the absence of effect of FCCP (100 and 300 nM) in a myocyte dialyzed with a pipette solution in which ATP was substituted with non-hydrolysable ATP analogues AMP-PCP (3 mM) and ATP-γ-S (0.5 mM). (B) Mean amplitudes of ICa,L recorded in control and with ISO (1 μM) during metabolic inhibition in cells dialyzed with 3 mM of ATP (white bars) or with non-hydrolysable ATP analogues (grey bars). *** P<0.001.

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  Fig 7. Effect of FCCP on ICa,L in cells dialyzed with zero-Mg2+ solution. Comparison of the response to ISO (1 μM) and to FCCP (30 and 100 nM) in the presence of ISO in the absence (white bars) or presence (black bars) of 4 mM Mg2+ in the pipette solution. *P<0.05.

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  Fig 8. Effect of intracellular pH on ICa,L response to metabolic inhibition. (A) Effect of 100 and 300 nM of FCCP on ISO (1 μM)-stimulated ICa,L in cells dialyzed with either control (pH 7.3, white bar) or alkaline solution (pH 8.3, black bars). * P<0.05. (B) (a) Effect of three different FCCP concentrations (30, 50 and 100 nM) on ISO (1 μM)-stimulated ICa,L in a ventricular myocyte dialysed with low HEPES (1 mM) intracellular solution. (b) Concentration-response curves for the effects of FCCP on ISO-stimulated ICa,L in cells dialysed with a solution containing either 1 mM (n = 3 to 7, white squares) or 10 mM HEPES (n = 4, black diamonds).

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  Fig 9. Schematic representation of the metabolic inhibition of cardiac ICa,L. Metabolic inhibition leads to the depolarization of mitochondrial membrane potential. Under these conditions the mitochondrial F1F0-ATP synthase operates in the reverse-mode and intensive ATP hydrolysis results in intracellular acidification which in turn reduces ICa,L.