Distinct localization and modulation of Cav1.2 and Cav1.3 L-type Ca2+ channels in mouse sinoatrial node

Abstract

Dysregulation of L-type Ca(2+) currents in sinoatrial nodal (SAN) cells causes cardiac arrhythmia. Both Ca(v)1.2 and Ca(v)1.3 channels mediate sinoatrial L-type currents. Whether these channels exhibit differences in modulation and localization, which could affect their contribution to pacemaking, is unknown. In this study, we characterized voltage-dependent facilitation (VDF) and subcellular localization of Ca(v)1.2 and Ca(v)1.3 channels in mouse SAN cells and determined how these properties of Ca(v)1.3 affect sinoatrial pacemaking in a mathematical model. Whole cell Ba(2+) currents were recorded from SAN cells from mice carrying a point mutation that renders Ca(v)1.2 channels relatively insensitive to dihydropyridine antagonists. The Ca(v)1.2-mediated current was isolated in the presence of nimodipine (1 μm), which was subtracted from the total current to yield the Ca(v)1.3 component. With strong depolarizations (+80 mV), Ca(v)1.2 underwent significantly stronger inactivation than Ca(v)1.3. VDF of Ca(v)1.3 was evident during recovery from inactivation at a time when Ca(v)1.2 remained inactivated. By immunofluorescence, Ca(v)1.3 colocalized with ryanodine receptors in sarcomeric structures while Ca(v)1.2 was largely restricted to the delimiting plasma membrane. Ca(v)1.3 VDF enhanced recovery of pacemaker activity after pauses and positively regulated pacemaking during slow heart rate in a numerical model of mouse SAN automaticity, including preferential coupling of Ca(v)1.3 to ryanodine receptor-mediated Ca(2+) release. We conclude that strong VDF and colocalization with ryanodine receptors in mouse SAN cells are unique properties that may underlie a specific role for Ca(v)1.3 in opposing abnormal slowing of heart rate.

Figure 1. Pharmacological isolation of I_Ba mediated by Ca_v1.2 and Ca_v1.3 in Ca_v1.2 DHP mouse sinoatrial node (SAN) cells

A, phase contrast image of a representative mouse SAN cell used for whole cell patch-clamp recordings. B, effects of nimodipine (NIM, 1 μm) on I_Ba in wild-type (WT, n = 3) mouse SAN cells. C, same as in B except with Ca_v1.2 DHP SAN cells (n = 23). The residual current in the presence of NIM (Ca_v1.2) was subtracted from the total current to yield the Ca_v1.3 component. In B and C, representative I_Ba traces and voltage protocol (left) and current–density (pA pF⁻¹) vs. voltage (mV) relation (right) are shown.

Figure 2. Ca_v1.3 currents activate faster and inactivate more slowly than Ca_v1.2 currents in mouse sinoatrial node cells

A, Ca_v1.2 and Ca_v1.3-mediated I_Ba was evoked and isolated as in Fig. 1B. Current traces (left) were fit with a single exponential function (smooth black line). The resulting time constants (τ) were plotted against test voltage (right, n = 22). B, inactivation of I_Ba evoked by 500 ms test pulses to a test voltage of −10 mV. I_res/I_peak = current amplitude at the end of the pulse normalized to peak amplitude, plotted for test voltages of −30 and −10 mV. *P = 0.01, one-way ANOVA, n = 8.

Figure 3. Ca_v1.3 and Ca_v1.2 undergo VDI in mouse sinoatrial node (SAN) cells

Top, voltage protocol. Test currents were evoked by 50 ms test pulses before (P1) and after (P2) a 200 ms conditioning prepulse. Middle, representative I_Ba evoked by P1 (black trace) and P2 (grey trace). Bottom, fractional current (P2/P1) was plotted against prepulse voltage. *P < 0.001, two-way ANOVA for group and voltage, Tukey post hoc test, n = 9.

Figure 4. Ca_v1.3 but not Ca_v1.2 undergoes voltage-dependent facilitation during recovery from inactivation

Top, voltage protocol. Test currents were evoked by 50 ms test pulses before (P1) and at variable intervals (Δt) after (P2) a 200 ms conditioning prepulse to +80 mV. Middle, representative I_Ba evoked by P1 (black trace) and P2 (grey trace). Bottom, fractional current was plotted against prepulse to P2 interval. *P < 0.01, #P < 0.001 for Ca_v1.3 vs. Ca_v1.2 by one-way anova and Tukey post hoc test, n = 8.

Figure 5. Confocal micrographs showing double labelling of RYR2 and Ca_v1.2 (A–E) or Ca_v1.3 (F–J)

Regions of colocalization are yellow in the merged images (C, D, H and I). E and J, line scan analysis through region marked with dashed lines in D and I showing signals for RYR2 (red) and Ca_v1.2 or Ca_v1.3 (green).

Figure 6. Numerical modelling of VDF in mouse sinoatrial node cells

A and B, fitting by the model of P2/P1 ratio as a function of the conditioning prepulse voltage (A) or recovery interval (B). Voltage protocols were the same as in Figs. 3 and 4. C, simulation of voltage-clamp experiments showing recovery from inactivation of Ca_v1.3-mediated I_Ca,L with (grey line) or without (black line) voltage-dependent facilitation. Dashed lines indicate zero current level and maximal I_Ca,L during recovery from inactivation. For the voltage-clamp protocol (left), the 200 ms conditioning prepulse was set to +30 mV.

Figure 7. Effects of Ca_v1.3 voltage-dependent facilitation (VDF) on I_Ca,L and sinoatrial node (SAN) cell pacemaking in a mathematical model

A, numerical simulations, including Ca_v1.3 VDF of SAN action potentials (APs), I_Ca,L (Ca_v1.3, black line; Ca_v1.2-mediated, grey line), I_Kr, and I_f. B, comparison between pacemaker activity (APs), Na⁺/Ca²⁺ exchanger current (I_NCX), subsarcolemmal Ca²⁺ concentration (SS_Ca), and SR Ca²⁺ release (SR_Ca) in our model. C, Ca_v1.3-mediated I_Ca,L calculated with VDF (grey line) or without VDF (black line). D, corresponding pacemaker activity with VDF (grey line) or without VDF (black line).

Figure 8. Activation of I_KATP transiently stopped automaticity in a model including VDF (A) and excluding VDF (B)

Arrows indicate the onset of I_KATP activation. Pacemaking resumed after pause. At steady state, pacemaker activity was slowed by 18% in the model including VDF and by 20% in the model excluding VDF. C and D show corresponding Ca_v1.3-mediated I_Ca,L, while dashed arrows indicate Ca_v1.3-mediated I_Ca,L at the first beat upon resuming of automaticity. E and F, enlargement of Ca_v1.3-mediated I_Ca,L at the first beat and steady state upon resuming of automaticity in the model including (grey line) and excluding VDF (black line). Traces represent superimposed Ca_v1.3-mediated I_Ca,L during steady-state pacemaker activity after I_KATP activation in the model including (grey line) and excluding VDF (black line). Steady-state pacemaker activity (G) and Ca_v1.3-mediated I_Ca,L (H) after activation of I_KATP in the model including VDF (grey line) or after abolition of VDF (black line).