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

1. In the present study Ca2+ entry via different voltage-dependent membrane channels was examined with a fluorescent Ca2+ indicator before and after b eta-adrenergic stimulation. 2. To clearly distinguish between Ca2+ influx and Ca2+ release from the sar coplasmic reticulum the Ca2+ store was blocked with 0.1 mu M thapsigargin a nd 10 mu M ryanodine. Omitting Nat from the pipette filling solution minimi zed Ca2+ entry via Na+-Ca2+ exchange. 3. Individual guinea-pig ventricular myocytes were voltage clamped in the w hole-cell configuration of the patch-clamp technique and different membrane currents were activated using specific voltage protocols. The intracellula r Ca2+ concentration was simultaneously recorded with a laser-scanning conf ocal microscope using fluo-3 as a Ca2+ indicator. 4. Ca2+ entry pathways were discriminated using pharmacological blockers un der control conditions and during beta-adrenergic stimulation with 1 mu M i soproterenol (isoprenaline) in the bathing solution or 100 mu M cAMP in the patch-clamp pipette. 5. Isoproterenol or cAMP potentiated the Ca2+ influx signals recorded durin g L-type Ca2+ current activation but, more interestingly, also during Na+ c urrent (I-Na) activation. The Ca2+ influx signal arising from L-type Ca2+ c urrent activation was usually blocked by 50 mu M Cd2+. However, the Ca2+ in flux signal elicited by the Na+ current activation protocol was only curtai led to 56.4 +/- 28.2% by 100 mu M Ni2+ but was reduced to 17.9 +/- 15.1% by 50 mu M Cd2+ and consistently eliminated by 5 mM Ni26. The pronounced Cd2+ and moderate Ni2+ sensitivity of the Ca2+ influx sig nals suggested that the predominant source of Ca2+ influx during the Na+ cu rrent activation - before and during beta-adrenergic stimulation - was a sp urious activation of the L-type Ca2+ current, presumably due to voltage esc ape during Na+ current activation. 7. Calculations based on the relationship between Ca2+ current and fluoresc ence change revealed that, on average, we could reliably detect rapid Ca2concentration 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 N a+ channels which is less than 0.04:1 for Ca2+ ions flowing through Na+ cha nnels via the proposed 'slip-mode' Ca2+ conductance. Therefore the slip-mod e Ca2+ conductance of Na+ channels does not contribute noticeably to the Ca 2+ signals observed in our experiments.