L‐type Ca 2+ current as the predominant pathway of Ca 2+ entry during I Na activation in β‐stimulated cardiac myocytes

1 In the present study Ca 2+ entry via different voltage‐dependent membrane channels was examined with a fluorescent Ca 2+ indicator before and after β‐adrenergic stimulation. 2 To clearly distinguish between Ca 2+ influx and Ca 2+ release from the sarcoplasmic reticulum the Ca 2+ store was blocked with 0.1 μ m thapsigargin and 10 μ m ryanodine. Omitting Na + from the pipette filling solution minimized Ca 2+ entry via Na + ‐Ca 2+ 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 Ca 2+ concentration was simultaneously recorded with a laser‐scanning confocal microscope using fluo‐3 as a Ca 2+ indicator. 4 Ca 2+ entry pathways were discriminated using pharmacological blockers under control conditions and during β‐adrenergic stimulation with 1 μ m isoproterenol (isoprenaline) in the bathing solution or 100 μ m cAMP in the patch‐clamp pipette. 5 Isoproterenol or cAMP potentiated the Ca 2+ influx signals recorded during L‐type Ca 2+ current activation but, more interestingly, also during Na + current ( I Na ) activation. The Ca 2+ influx signal arising from L‐type Ca 2+ current activation was usually blocked by 50 μ m Cd 2+ . However, the Ca 2+ influx signal elicited by the Na + current activation protocol was only curtailed to 56.4 ± 28.2 % by 100 μ m Ni 2+ but was reduced to 17.9 ± 15.1 % by 50 μ m Cd 2+ and consistently eliminated by 5 m m Ni 2+ . 6 The pronounced Cd 2+ and moderate Ni 2+ sensitivity of the Ca 2+ influx signals suggested that the predominant source of Ca 2+ influx during the Na + current activation – before and during β‐adrenergic stimulation – was a spurious activation of the L‐type Ca 2+ current, presumably due to voltage escape during Na + current activation. 7 Calculations based on the relationship between Ca 2+ current and fluorescence change revealed that, on average, we could reliably detect rapid Ca 2+ concentration changes as small as 5.4 ± 0.7 n m . Thus, we can estimate an upper limit for the Ca 2+ permeability of the phosphorylated TTX‐sensitive Na + channels which is less than 0.04:1 for Ca 2+ ions flowing through Na + channels via the proposed ‘slip‐mode’ Ca 2+ conductance. Therefore the slip‐mode Ca 2+ conductance of Na + channels does not contribute noticeably to the Ca 2+ signals observed in our experiments.