L-type Ca2+ current as the predominant pathway of Ca2+ entry during I(Na) activation in beta-stimulated cardiac myocytes.
In the present study Ca2+ entry via different voltage-dependent membrane channels was examined with a fluorescent Ca2+ indicator before and after beta-adrenergic stimulation. To clearly distinguish between Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum the Ca2+ store was blocked with 0.1 microM thapsigargin and 10 microM ryanodine. Omitting Na+ from the pipette filling solution minimized Ca2+ entry via Na+-Ca2+ exchange. 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. Ca2+ entry pathways were discriminated using pharmacological blockers under control conditions and during beta-adrenergic stimulation with 1 microM isoproterenol (isoprenaline) in the bathing solution or 100 microM cAMP in the patch-clamp pipette. Isoproterenol or cAMP potentiated the Ca2+ influx signals recorded during L-type Ca2+ current activation but, more interestingly, also during Na+ current (INa) activation. The Ca2+ influx signal arising from L-type Ca2+ current activation was usually blocked by 50 microM Cd2+. However, the Ca2+ influx signal elicited by the Na+ current activation protocol was only curtailed to 56.4 +/- 28.2 % by 100 microM Ni2+ but was reduced to 17.9 +/- 15.1 % by 50 microM Cd2+ and consistently eliminated by 5 mM Ni2+. 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 beta-adrenergic stimulation - was a spurious activation of the L-type Ca2+ current, presumably due to voltage escape during Na+ current activation. 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.
DelPrincipe
F
F
Department of Physiology, University of Bern, Buhlplatz 5, CH-3012 Bern, Switzerland.
Egger
M
M
Niggli
E
E
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000465:Algorithms; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D006321:Heart / Q000187:drug effects; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018613:Microscopy, Confocal; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D012433:Ryanodine / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
D000318:Adrenergic beta-Agonists; D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D015222:Sodium Channels; D012433:Ryanodine; D019284:Thapsigargin; D002118:Calcium