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

Abstract

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.

Figure 1. Simultaneous recording of inward current and Ca²⁺ release

A, activation of the L-type Ca²⁺ current (200 ms) elicited a substantial Ca²⁺ release signal. Both the current and the Ca²⁺ signal were markedly enhanced by administration of 1 μm isoproterenol in the bathing solution. B, in an analogous experiment, activation of the Na⁺ current (50 ms) in a different cell showed smaller Ca²⁺ signal amplitudes but similar β-adrenergic potentiation. Traces show from top to bottom: voltage protocol (mV), current record, line-scan image, mean Ca²⁺ concentration profile, same current record expanded 10 times (red traces, not temporally aligned to the other signals).

Figure 2. Simultaneous recording of inward current and Ca²⁺ influx

SR Ca²⁺ release was blocked by treating the cells with 0.1 μm thapsigargin and 10 μm ryanodine. Na⁺ current was activated in the presence of 1 μm isoproterenol. A, activation of L-type Ca²⁺ current elicited an detectable Ca²⁺ influx signal with reduced amplitude due to the blockade of the SR release (note the absence of sparks). A potentiating effect of isoproterenol on the current amplitude and the Ca²⁺ signal is evident. B, analogous experiment with activation of the Na⁺ current. Note the change in the scale and the small amplitude of the Ca²⁺ signals. Traces are arranged in the same way as in Fig. 1.

Figure 3. Pharmacological identification of the Ca²⁺ influx pathway

All cells were treated with 0.1 μm thapsigargin and 10 μm ryanodine. Na⁺ current was activated in the presence of 1 μm isoproterenol. A, the administration of 100 μm Ni²⁺ failed to block the Ca²⁺ influx signal. However, a slight reduction of the Ca²⁺ signal amplitude is noticeable. B, the administration of 5 mm Ni²⁺ blocked the Ca²⁺ signal completely. C, the Ca²⁺ influx signal was eliminated by 50 μm Cd²⁺. Traces are arranged in the same way as in Fig. 1. D, normalized Ca²⁺ influx amplitudes versus different concentrations of Ni²⁺ and of 50 μm Cd²⁺. Data are plotted as means ±s.d. The inset shows a Hill function fitted to the data points of the Ni²⁺ block. The calculated IC₅₀ was 114 ± 33.2 μm (95% confidence); the Hill coefficient was 1.71. For comparison, the Hill function of the Ni²⁺ dose-response curves of the L-type current in guinea-pig ventricular myocytes (blue trace, taken from Hobai et al. 1998) and in the human heart T-type α1H subunit (T-type, red trace, taken from Lee et al. 1999) have been added.

Figure 4. Ca²⁺ influx under elevated cAMP and blockade by TTX

A, with 100 μm cAMP in the pipette filling solution, activation of I_Na elicited a Ca²⁺ influx signal which was blocked by 5 mm Ni²⁺. Note the increase in fluorescence in the right line-scan, which is due to elevated extracellular Ca²⁺ (10 mm). B, the Na⁺ current and the Ca²⁺ influx signal elicited in the presence of 1 μm isoproterenol were completely blocked by 20 μm TTX.

Figure 5. Forced Ca²⁺ influx under extreme conditions

SR release was blocked and the Na⁺ current was activated repetitively in the presence of 1 μm isoproterenol. A, a Ca²⁺ influx signal could be elicited by a train of 50 depolarizing pulses at 33 Hz. The Ca²⁺ influx signal was eliminated by 5 mm Ni²⁺. B, an analogous experiment was performed in the absence of isoproterenol but with 100 μm cAMP in the pipette filling solution. Sodium currents were activated repetitively 100 times at 33 Hz.

Figure 6. Estimation of the detection limit and relationship to a calculated permeability ratio

A, in a cell with blocked SR CICR an L-type Ca²⁺ current and the resulting Ca²⁺ influx signal were recorded simultaneously under control conditions. B, the Cd²⁺-sensitive difference current was integrated and plotted against the rising phase of the Ca²⁺ signal. The fitted line relates the observed Ca²⁺ influx fluorescence amplitude to the corresponding Ca²⁺ current in this given cell and corresponds to a cytosolic buffer capacity of about 28 (note that the SR was blocked). C, an mean Ca²⁺ concentration profile was plotted from a line-scan image and the standard deviation of the noise was calculated. After 500 ms, a step increase in Ca²⁺ was simulated by adding a concentration jump corresponding to a multiple of the s.d. The first step that is clearly visible coincides with +3 s.d. and, for this particular cell, corresponds to a concentration change of ∼8 nm[Ca²⁺]_i. Taking the relationship illustrated in B we can estimate our mean detection limit for Ca²⁺ influx to be around 2 pC (dashed horizontal line in D). D, using the Goldmann-Hodgkin-Katz equation, the permeability ratio P_Ca/P_Na was calculated for a 50 nA (□, ▪) or 10 nA Na⁺ current (○, •) and for 10 mm (•, ▪) or 1 mm[Ca²⁺]_o (○, □), respectively.