CaV1.3 L-type Ca2+ channel contributes to the heartbeat by generating a dihydropyridine-sensitive persistent Na+ current

Figure 1. Presence of I st in mouse SAN cells. (A) (a) Superimposed whole-cell membrane currents recorded from the same cell in 1.8 mM [Ca2+]o (control, black), in 0.1 mM [Ca2+]o (blue) and after exposure to 1 µM isradipine (red). Individual SAN cells were voltage clamped at a holding potential of −90 mV and depolarized for 1 s to indicated test potentials in 10-mV increments. Peaks of transient inward currents at the beginning of test pulses are not to scale. The inset shows close-up views of the initial part (red shaded area) of current traces. (b) Corresponding isochronal I–V relationships of the late current measured at time points marked with arrows in a. (c) I–V relationship of the isradipine-sensitive current obtained by subtraction of the current recorded after application of isradipine from that recorded in 0.1 mM [Ca2+]o. (B) (a) Superimposed whole-cell membrane currents recorded in the same cell in 1.8 mM [Ca2+]o (control, black), in NMDG-substituted, Na+-free solution (blue) and after exposure to 1 µM nifedipine (red) using the same pulse protocol as in (A). The inset shows close-up views of the initial part (red shaded area) of current traces. (b) Corresponding I–V…

Figure 2. Pharmacological properties of I st in mouse SAN cells. (A) Effects of TTX on I st. (a) Voltage ramp pulse protocol and original current traces recorded from the same cell in 0.1 mM [Ca2+]o (control, black), during exposure to 10 µM TTX (blue) and after subsequent addition of 1 µM nifedipine (red). The inset shows expanded traces at the beginning of the voltage-command pulse. (b) Corresponding I–V relationship obtained from current recordings during the descending ramp from + 40 to −110 mV in a. (c) I–V relationship of I st isolated by subtracting current recordings before and after application of nifedipine in the presence of TTX. (B) Effects of Na+ replacement with Li+ on I st. The I–V relationships were constructed on current recordings before (black) and after (blue) total replacement of Na+ with Li+ in the 0.1 mM [Ca2+]o solution. Currents were elicited by 1-s depolarizing pulses (10 mV increment) to various test potentials from a holding potential of −90 mV. The inset shows original current traces at −50 mV. (C) Effects of autonomic agonists on I st. The I–V relationships were obtained from current recordings in the same cell during the descending limb of a voltage ramp…

Figure 3. Absence of I st in SAN cells from CaV1.3−/− mice. (A) Representative examples of current recordings in SAN cells obtained from wild-type (upper panel) and CaV1.3−/− (lower panel) mice. Currents were elicited by voltage steps to test potentials between −80 and + 40 mV (10 mV increments) preceded by a conditioning pulse to −50 mV from a holding potential of −90 mV in 1.8 mM [Ca2+]o (control, black), in 0.1 mM [Ca2+]o solution containing 10 µM TTX (blue) and after applying 1 µM nifedipine (red). (B) Average I–V relationships of current densities measured at the time points indicated in (A). Data represent the mean ± S.E.M. of wild-type (left, n = 19) and CaV1.3−/− (right, n = 18) SAN cells. (C), I–V relationships of I Ca,L in wild-type (closed symbols) and CaV1.3−/− (open symbols) SAN cells, obtained by subtraction of currents after lowering [Ca2+]o from recordings in the control solution. (D) I–V relationships of I st in wild-type (closed symbols) and CaV1.3−/− (open symbols) SAN cells, measured as the nifedipine-sensitive current in 0.1 mM [Ca2+]o.

Figure 4. CaV1.3 mediates both I Ca,L and I st in the SAN diastolic depolarization range. (A,C) Representative whole-cell membrane currents recorded from the same wild-type SAN cell using two different external solutions: TEA+-substituted, Na+-free external solution containing 1.8 mM Ca2+ (A) to record I Ca,L and 140 mM [Na+]o solution containing 0.1 mM Ca2+ plus 10 µM TTX (C) to record I st. The cell was first held at −75 mV. Then, a slow ascending ramp (100 mV/s) voltage command was used to elicit I Ca,L or I st (top panel), from −65 to −35 mV, followed by depolarization to +10 mV for 50 ms. Under distinct external conditions, currents were recorded in the absence (black trace, left panel) and presence (blue trace, left panel) of 100 nM Iso, and after addition of 1 µM nifedipine (red trace, left panel). Nifedipine-sensitive net I Ca,L and I st in the absence (black trace, right panel) and presence (blue trace, right panel) of Iso were obtained by digital subtraction of current traces before and after application of nifedipine (right panel). (B,D) Representative I Ca,L (B) and I st (D) in CaV1.3−/− SAN cells, recorded using the same protocol as in (A,C), respectively. (E) Sample current…

Figure 5. Sensitivity of I Ca,L and I st to nifedipine in SAN cells from CaV1.2DHP−/− mice. (A) I Ca,L inhibition by nifedipine in CaV1.2DHP−/− SAN cells. (a) Superimposed sample traces of I Ca,L elicited by depolarization to −10 mV from a holding potential of −50 mV in TEA+-substituted, Na+-free Tyrode solution (control, black) and during nifedipine application at various concentrations (0.03–1 µM, red), and after subsequent addition of 3 µM verapamil (blue). (b) Concentration-dependent inhibition of I Ca,L by nifedipine. Inhibition is expressed as the percentage of residual I Ca,L inhibited by nifedipine relative to control I Ca,L. Data are mean ± S.E.M. of four experiments. The smooth curve represents the least squares fit of data points using the Hill equation, yielding a maximal current response of 72%, IC50 of 101.7 nM and Hill coefficient of 0.97. (B) Effects of nifedipine on I st in wild-type and CaV1.2DHP−/− SAN cells. (a) Superimposed I–V relationships of I st obtained by voltage ramp in wild-type (left) and CaV1.2DHP−/− (right) SAN cells in the control 0.1 mM [Ca2+]o solution (control, black), during exposure to various concentrations of nifedipine (0.03–1 µM, red), and after…