A bilobal model of Ca2+-dependent inactivation to probe the physiology of L-type Ca2+ channels

Figures

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  Figure 1.. Using photo-uncaging to quantify CDI. (A–D) CaV1.2E2A channels are coexpressed with mutant CaM1234 in HEK293 cells to characterize pore block. (A) Li+ currents before Ca2+ uncaging (black) with simultaneously measured resting [Ca2+] (red). (B) UV photo-uncaging of Ca2+ (blue) results in a step increase in [Ca2+] (red) and minimal reduction of Li+ current (black) compared with preflash conditions (gray). (C) Li+ current recorded before (gray) and after (black) UV photo-uncaging of Ca2+. A higher-intensity UV flash results in a larger Ca2+ step (red), compared with B, yet there is a small decrease in current, indicating minimal pore block in this channel. (D) Steady-state pore block as a function of [Ca2+], fitted with a Hill coefficient of 1 and large Kd of 33 µM (black curve). Data are from 13 replicates (variable level UV flashes) across 7 cells. Error bars indicate ±SEM. (E–H) CDI from CaV1.2E2A coexpressed with CaMWT. (E) Li+ current (black) at resting [Ca2+] (red; F) UV flash produces a large Ca2+ step (red) and concurrent reduction in current (black) indicative of CDI. Gray trace shows preflash current. (G) Fractional block of current after UV flash as a function of [Ca2+].…

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  Figure 2.. Quantification of CaM lobe-specific CDI. (A–C) CaV1.2E2A channels are coexpressed with mutant CaM34 in HEK293 cells, so as to characterize N-lobe CDI. (A) Li+ currents before Ca2+ uncaging (black) with simultaneously measured resting [Ca2+] (red). (B) UV photo-uncaging of Ca2+ (blue) results in a step increase in [Ca2+] (red) and moderate reduction of Li+ current (black) compared with preflash conditions (gray). (C) Steady-state N-lobe CDI as a function of [Ca2+], fitted with a Hill coefficient of 1.8 and Kd of 0.9 µM. Data are from 19 replicates (variable level UV flashes) across nine cells. Error bars indicate ±SEM. (D–F) CDI from CaV1.2E2A coexpressed with CaM12. (D) Li+ currents before Ca2+ uncaging (black) with simultaneously measured resting [Ca2+] (red). (E) UV photo-uncaging of Ca2+ (blue) results in a step increase in [Ca2+] (red) and moderate reduction of Li+ current (black) compared with preflash conditions (gray). (F) Steady-state C-lobe CDI as a function of [Ca2+], fitted with a Hill coefficient of 1.4 and Kd of 1.15 µM. Data are from 21 replicates (variable level UV flashes) across 13 cells. Error bars indicate ±SEM.

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  Figure 3.. Kinetic model of CDI mediated by N-lobe CaM. (A) Schematic representation of N-lobe–mediated CDI model of CaV1.2 channels. Rate constants are shown above and below the arrows depicting the state transition (Table 1). (B) Simulated ICa(L) (red) in response to three different levels of Ca2+ recapitulates the N-lobe CDI recorded from CaV1.2E2A coexpressed with CaM34 (black). Ca2+ step (red) is idealized (see text) from experimental data and used as the model input. (C) Steady-state N-lobe–mediated CDI predicted by this four-state N-lobe model (red) matches well to the experimental data (black, taken from Fig. 2 C as reference). Error bars indicate ±SEM.

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  Figure 4.. Kinetic model of CDI mediated by C-lobe CaM. (A) Schematic representation of C-lobe–mediated CDI model of CaV1.2 channels. Rate constants are shown above and below the arrows depicting state transitions (Table 1). (B) Simulated ICa(L) (red) in response to three different levels of Ca2+ recapitulates the C-lobe CDI recorded from CaV1.2E2A coexpressed with CaM12 (black). Ca2+ step (red) is idealized (see text) from experimental data and used as the model input. (C) Steady-state C-lobe–mediated CDI predicted by this four-state C-lobe model (red) matches well to the experimental data (black, taken from Fig. 2 F as reference).

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  Figure 5.. Full bilobal model of CDI. (A) Schematic representation of 16-state CaM-mediated CaV1.2 CDI. Rate constants are shown above and below the arrows depicting the state transition (Table 1). (B) Simulated ICa(L) (red) in response to three different levels of Ca2+ recapitulates the CDI recorded from CaV1.2E2A coexpressed with CaMWT (black). Ca2+ step (red) is idealized (see text) from experimental data and used as the model input. (C) Steady-state CaM-mediated CaV1.2 CDI predicted by this 16-state bilobal model (red) matches well to the experimental data (black, taken from Fig. 1 H as reference). Error bars indicate ±SEM.

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  Figure 6.. Full bilobal model of CDI in the context of a calmodulinopathic mutation. (A) Disruption of action potentials in guinea pig ventricular myocytes by CaMD130G. When CaMD130G was overexpressed, cardiac action potentials become markedly lengthened and irregular (red) compared with CaMWT (shown in gray). Adapted from Limpitikul et al. (2014). (B) Simulated ICa(L) (red) in response to three different levels of Ca2+ recapitulates the CDI recorded from CaV1.2E2A coexpressed with CaMD130G (black). Ca2+ step (red) is idealized (see text) from experimental data and used as the model input. (C) Steady-state CaM-mediated CaV1.2 CDI predicted by the adjusted 16-state bilobal model (red) matches well to the experimental data (black). Data are from 18 replicates across eight cells. Error bars indicate ±SEM.