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L-type Ca²⁺ current as the predominant pathway of Ca²⁺ entry during I_Na activation in -stimulated cardiac myocytes

Franco DelPrincipe, Marcel Egger and Ernst Niggli

1. In the present study Ca²⁺ entry via different voltage-dependent membrane channels was examined with a fluorescent Ca²⁺ indicator before and after -adrenergic stimulation.

2. To clearly distinguish between Ca²⁺ influx and Ca²⁺ release from the sarcoplasmic reticulum the Ca²⁺ store was blocked with 0·1 µM thapsigargin and 10 µM ryanodine. Omitting Na⁺ from the pipette filling solution minimized Ca²⁺ entry via Na⁺-Ca²⁺ exchange.

3. Individual guinea-pig ventricular myocytes were voltage clamped in the whole-cell configuration of the patch-clamp technique and different membrane currents were activated using specific voltage protocols. The intracellular Ca²⁺ concentration was simultaneously recorded with a laser-scanning confocal microscope using fluo-3 as a Ca²⁺ indicator.

4. Ca²⁺ entry pathways were discriminated using pharmacological blockers under control conditions and during -adrenergic stimulation with 1 µM isoproterenol (isoprenaline) in the bathing solution or 100 µM cAMP in the patch-clamp pipette.

5. Isoproterenol or cAMP potentiated the Ca²⁺ influx signals recorded during L-type Ca²⁺ current activation but, more interestingly, also during Na⁺ current (I_Na) activation. The Ca²⁺ influx signal arising from L-type Ca²⁺ current activation was usually blocked by 50 µM Cd²⁺. However, the Ca²⁺ influx signal elicited by the Na⁺ current activation protocol was only curtailed to 56·4 ± 28·2 % by 100 µM Ni²⁺ but was reduced to 17·9 ± 15·1 % by 50 µM Cd²⁺ and consistently eliminated by 5 mM Ni²⁺.

6. The pronounced Cd²⁺ and moderate Ni²⁺ sensitivity of the Ca²⁺ influx signals suggested that the predominant source of Ca²⁺ influx during the Na⁺ current activation - before and during -adrenergic stimulation - was a spurious activation of the L-type Ca²⁺ current, presumably due to voltage escape during Na⁺ current activation.

7. Calculations based on the relationship between Ca²⁺ current and fluorescence change revealed that, on average, we could reliably detect rapid Ca²⁺ concentration changes as small as 5·4 ± 0·7 nM. Thus, we can estimate an upper limit for the Ca²⁺ permeability of the phosphorylated TTX-sensitive Na⁺ channels which is less than 0·04:1 for Ca²⁺ ions flowing through Na⁺ channels via the proposed 'slip-mode' Ca²⁺ conductance. Therefore the slip-mode Ca²⁺ conductance of Na⁺ channels does not contribute noticeably to the Ca²⁺ signals observed in our experiments.

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