Role of the Ca_V1.2 distal carboxy terminus in the regulation of L-type current

Figures & data

Figure 1. DC_termD effect over Ca_V1.2^Δ1820: (a) Representative whole-cell L-type Ba²⁺ current traces from AD293 cells overexpressing the truncated version of Ca_V1.2 channel (Ca_V1.2^Δ1820) with or without the DC_termD. Currents elicited by a voltage step protocol from − 60 to +50 mV in 10-mV increments, V_h = −80 mV. (b) Summary peak current I/V plots (mean ± SEM) obtained from currents family as shown in (A); black lines represent the best fit to a Boltzmann equation. c) Graph shows the mean ± SEM of the peak Ba²⁺ current density. d) Graph shows the mean ± SEM of the midpoint of activation. In every panel, control cells are represented with filled circles and cells co-expressing the DC_termD with empty circles. *p < 0.05 with respect to control, n = 8 for control and n = 7 for DC_termD.

Description of the effect of DCtermD on CaV1.2Δ1820: A) Visual representation of whole-cell L-type Ba2+ current traces from AD293 cells overexpressing CaV1.2Δ1820 with or without the DCtermD. B) Summary peak current I/V plots (mean ± SEM) derived from the current traces shown in (A). Black lines indicate the best fit to a Boltzmann equation. C) Graph illustrating the mean ± SEM of the peak Ba2+ current density. D) Graph presenting the mean ± SEM of the midpoint of activation. In each panel, filled circles denote cells overexpressing the truncated version of CaV1.2 channel, while empty circles represent cells co-expressing DCtermD and CaV1.2Δ1820. *P < 0.05 compared to control.

Figure 2. DC_termD modifies VDI and CDI of Ca_V1.2^Δ1820: (a) Graph showing the residual of current after 200 ms during a 250-ms depolarization pulse (R₂₀₀, mean ± SEM, n = 8 for control and n = 7 for DC_termD) versus command voltage of L-type Ba²⁺ currents AD293 cells overexpressing the Ca_V1.2^Δ1820 channel with (empty circles) or without (filled circles) the DC_termD. Slower inactivation rates result in higher R₂₀₀ values. (b) Representative trace of the I_Ba (black line) and I_Ca evoked by a 0 mV depolarizing pulse. Both currents were scaled to the same amplitude for comparison. (c) Graph shows the mean ± SEM of CDI fraction (n = 5 for control and n = 6 for DC_termD). *p < 0.05 with respect to control.

Figure 3. DCRD peptide displaces DC_termD from Ca_V1.2^Δ1820 BRET titration curve from AD293 cells co-transfected with increasing amounts of Ca_V1.2^Δ1820-YFP with (gray circles) or without (black circles) the DCRD peptide coding sequence, and a constant amount of DC_termD-RLuc as the energy donor. White circles denote experiments were the DC_termD without the RLuc was used. Saturation of the BRET curve indicates specific interaction between the DC_termD and the channel pore, whereas the low linear BRET signal denoted absence of interaction. Dashed lines represent the best fit to a hyperbolic curve or a linear fit respectively. Data from five experiments are included in the graph.

Analysis of DCRD peptide impact on DCtermD displacement from CaV1.2Δ1820 Graph showing the net BRET versus the ratio of YFP and Cltz emitted light form cells overexpressing CaV1.2Δ1820-YFP and the DCtermD with or without the DRCD peptide. A saturation curve is observed only in the absence of the DRCD peptide.

Figure 4. Effect of the DCRD peptide over Ca_V1.2^Δ1820. (a) Representative whole-cell L-type Ba²⁺ current traces from AD293 cells overexpressing the Ca_V1.2^Δ1820 channel with or without the DCRD peptide. Currents elicited by a voltage step protocol from − 60 to +50 mV in 10-mV increments, V_h = −80 mV. (b) Summary peak current I/V plots (mean ± SEM) obtained from currents family as shown in (A); black lines represent the best fit to a Boltzmann equation. (c) Graph shows the mean ± SEM of the peak Ba²⁺ current density. (d) Graph shows the mean ± SEM of the midpoint of activation. In every panel, control cells are represented with filled circles and cells co-expressing the DCRD peptide with empty circles. Control data set is the same used in Figure 1. *p < 0.05 with respect to control, n = 8 for control and n = 7 for DCRD peptide.

Description of the effect of DCRD peptide overexpression on CaV1.2Δ1820: A) Visual representation of whole-cell L-type Ba2+ current traces from AD293 cells. B) Summary peak current I/V plots (mean ± SEM) derived from the current traces shown in (A). Black lines indicate the best fit to a Boltzmann equation. C) Graph illustrating the mean ± SEM of the peak Ba2+ current density. D) Graph presenting the mean ± SEM of the midpoint of activation. In each panel, filled circles denote cells overexpressing the truncated version of CaV1.2 channel, while empty circles represent cells co-expressing DCtermD and CaV1.2Δ1820. Control data set is the same used in Figure 1. *P < 0.05 compared to control.

Figure 5. No effect of DCRD peptide over Ca_V1.2^Δ1820 calcium-dependent inactivation: (a) and (c) Representative trace of Ca_V1.2^Δ1820 barium current (black line) and calcium current evoked by a 0 mV depolarizing pulses from control AD293 cells (a) or AD293 cells overexpressing calmodulin (c). I_Ba and I_Ca currents were scaled to the same amplitude for comparison. (b) and (d) Graph shows the mean ± SEM of CDI fraction from AD293 control cells (n = 5) (b) or AD293 cells overexpressing calmodulin (n = 8 for control, n = 6 for DC_termD, and n = 6 for DCRD peptide). The control data set of panel B is the same used in Figure 2c. *p < 0.05 with respect to control.

Impact of DCRD peptide on CaV1.2Δ1820 voltage-dependent inactivation: A) Graph depicting the residual current after 200 ms during a 250-ms depolarization pulse (R200) versus command voltage of L-type Ba2+ currents. B) and C) Graphs displaying the mean ± SEM of R200 in cells overexpressing CaM (panel C) or with endogenous CaM (panel B). Data from cells overexpressing CaV1.2Δ1820 alone or with the DCtermD, or the DCRD peptide is shown. The control dataset of panel A and B is identical to that used in Figure 2. *P < 0.05 compared to control.

Figure 6. The DCRD peptide modify Ca_V1.2^Δ1820 voltage-dependent inactivation: (a) Graph showing the residual of current after a 200-ms during a 250 ms depolarization pulse (R₂₀₀, mean ± SEM. n = 8 for control and n = 7 for DCRD peptide) versus command voltage of L-type Ba²⁺ currents from AD293 cells overexpressing the Ca_V1.2^Δ1820 channel with (empty circles) or without (filled circles) the DCRD peptide. Slower inactivation rates result in higher R₂₀₀ values. (b) Graph shows the mean ± SEM of R₂₀₀ from AD293 cells overexpressing the Ca_V1.2^Δ1820 channel alone (n = 5), or with the DC_termD (n = 6) or the DCRD peptide (n = 5). (c) Graph shows the mean ± SEM of R₂₀₀ from AD293 cells overexpressing calmodulin and the Ca_V1.2^Δ1820 channel alone (n = 8), or with the DC_termD (n = 7), or the DCRD peptide (n = 6). The control data set of panel a and B is the same used in Figure 2. *p < 0.05 with respect to control.

Impact of DCRD peptide on CaV1.2Δ1820 voltage-dependent inactivation: A) Graph depicting the residual current after 200 ms during a 250-ms depolarization pulse (R200) versus command voltage of L-type Ba2+ currents. B) and C) Graphs displaying the mean ± SEM of R200 in cells overexpressing CaM (panel C) or with endogenous CaM (panel B). Data from cells overexpressing CaV1.2Δ1820 alone or with the DCtermD, or the DCRD peptide is shown. The control dataset of panel A and B is identical to that used in Figure 2. *P*p <  compared to control.

Figure 7. DC_termD and DCRD peptide effect over endogenous L-type currents in cardiomyocytes (a) Graph shows the mean ± SEM of the peak endogenous L-type Ca²⁺ current density from newborn rat cardiomyocytes (n = 10 for control, n = 7 for DC_termD, and n = 7 for DCRD peptide). (b) Graph shows the mean ± SEM of R₂₀₀ newborn rat cardiomyocytes infected with RFP as control, or with the DC_termD-CFP, or the DCRD peptide coding sequence (n = 10 for control, n = 7 for DC_termD, and n = 7 for DCRD peptide). (c) Representative trace of the I_Ba (black line) and I_Ca evoked by a 0-mV depolarizing pulse. Both currents were scaled to the same amplitude for comparison. (d) Graph shows the mean ± SEM of CDI fraction (n = 7). *p < 0.05 with respect to control.

Impact of DCtermD and DCRD peptide on endogenous L-type currents in cardiomyocytes: A) Graph displaying the mean ± SEM of peak endogenous L-type Ca2+ current density from newborn rat cardiomyocytes. B) Graph depicting the residual L-type current after 200 ms during a 250-ms depolarization pulse (R200) from newborn rat cardiomyocytes. C) Illustrative trace of IBa (black line) and ICa evoked by a 0-mV depolarizing pulse. Both currents were adjusted to the same amplitude for comparison. D) Graph showing the mean ± SEM of calcium-dependent inactivation (CDI) fraction. Data of cardiomyocytes overexpressing RFP (as control), the DCtermD or the coding sequence of the DCRD peptide is shown. *p < 0.05 compared to control.

Data availability statement

The data that support the findings of this study are available from the corresponding author, DV, upon reasonable request.