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"Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA).

Zhu L, McDavid S, Currie KP - PLoS ONE (2015)

Bottom Line: The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA) had no effect.This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC.Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
CaV2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

No MeSH data available.


Related in: MedlinePlus

Recovery of CaV2 channels from “slow” inactivation: dependence on voltage and CaV β subunit, but not charge density.(A) Representative IBa from HEK293 cells transfected with CaV2.2, α2δ, and either β1b or β2a subunits. In both cases inactivation was almost complete by the end of the 10s step, but the β2a subunit dramatically reduced “fast” inactivation. The right panel shows the initial ~600ms of the traces on an expanded time base. (B) Recovery rate was not correlated with barium entry. Inactivation was produced by a 10s prepulse and recovery tracked as in Fig 1F. The recovery time constant (tau) is plotted against charge density (i.e. the integral of IBa during the 10s prepulse normalized to cell capacitance. The solid line shows a linear fit using Deming regression. The slope of this line was not significantly different from zero (p = 0.75 for β1b expressing cells and p = 0.76 for β2a expressing cells). (C) Recovery from inactivation was voltage-dependent. Inactivation of β2a containing channels was produced by a 10s prepulse and recovery tracked at different holding potentials (-80 mV, -100 mV, or -120 mV). Solid lines show exponential fits to the mean data (-120mV holding A = 0.79, t = 24.8 s; -100mV holding A = 0.8, t = 54.7 s; -80 mV holding A = 0.62, t = 103.2 s). The bar graph plots the mean time constant calculated from fits to the individual cells (* p < 0.05, *** p < 0.001 using ANOVA and Tukey’s post-test for pairwise comparisons).
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pone.0134117.g002: Recovery of CaV2 channels from “slow” inactivation: dependence on voltage and CaV β subunit, but not charge density.(A) Representative IBa from HEK293 cells transfected with CaV2.2, α2δ, and either β1b or β2a subunits. In both cases inactivation was almost complete by the end of the 10s step, but the β2a subunit dramatically reduced “fast” inactivation. The right panel shows the initial ~600ms of the traces on an expanded time base. (B) Recovery rate was not correlated with barium entry. Inactivation was produced by a 10s prepulse and recovery tracked as in Fig 1F. The recovery time constant (tau) is plotted against charge density (i.e. the integral of IBa during the 10s prepulse normalized to cell capacitance. The solid line shows a linear fit using Deming regression. The slope of this line was not significantly different from zero (p = 0.75 for β1b expressing cells and p = 0.76 for β2a expressing cells). (C) Recovery from inactivation was voltage-dependent. Inactivation of β2a containing channels was produced by a 10s prepulse and recovery tracked at different holding potentials (-80 mV, -100 mV, or -120 mV). Solid lines show exponential fits to the mean data (-120mV holding A = 0.79, t = 24.8 s; -100mV holding A = 0.8, t = 54.7 s; -80 mV holding A = 0.62, t = 103.2 s). The bar graph plots the mean time constant calculated from fits to the individual cells (* p < 0.05, *** p < 0.001 using ANOVA and Tukey’s post-test for pairwise comparisons).

Mentions: Given that PMA appeared to target recovery from “slow” but not “fast” inactivation, we investigated the effects of PMA on channels containing the β2a subunit which dramatically reduces fast inactivation [20, 21]. HEK293 cells were transiently transfected with CaV2.2, α2δ, and either β2a or β1b subunits (with EGFP as a marker). As expected, inactivation of IBa during a step depolarization was much slower in β2a than β1b containing channels (Fig 2A). Recovery of β2a channels was fit with a single exponential with a time constant (at a holding potential of -100 mV) of 48.6 ± 4.7 s (n = 16), which was significantly longer compared β1b containing channels (19.5 ± 1.8 s; n = 4; p = 0.0007 unpaired t-test). As expected for voltage-dependent inactivation, the recovery kinetics of IBa showed no correlation with the amount of barium entry (Fig 2B). The overall amount of barium entry (charge density) was much greater in cells expressing the β2a subunit than in G1A1 cells (β1b expressing cells). However, when the recovery time constant was plotted against charge density, the slope of a linear fit using Deming regression was not significantly different from zero (0 = 0.75 for β1b expressing cells and p = 0.76 for β2a expressing cells). This was confirmed using Pearson’s correlation coefficient which again showed no statistically significant correlation between recovery rate and charge density: for β1b cells r = -0.13, p = 0.74; for β2a cells r = -0.08, p = 0.76. We also found that, as predicted for voltage-dependent inactivation, recovery was significantly accelerated at more hyperpolarized holding potentials (Fig 2C). Thus, recovery following the conditioning prepulse demonstrated the expected features for voltage-dependent inactivation, and the recovery kinetics were influenced by the subtype of the CaVβ subunit.


"Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA).

Zhu L, McDavid S, Currie KP - PLoS ONE (2015)

Recovery of CaV2 channels from “slow” inactivation: dependence on voltage and CaV β subunit, but not charge density.(A) Representative IBa from HEK293 cells transfected with CaV2.2, α2δ, and either β1b or β2a subunits. In both cases inactivation was almost complete by the end of the 10s step, but the β2a subunit dramatically reduced “fast” inactivation. The right panel shows the initial ~600ms of the traces on an expanded time base. (B) Recovery rate was not correlated with barium entry. Inactivation was produced by a 10s prepulse and recovery tracked as in Fig 1F. The recovery time constant (tau) is plotted against charge density (i.e. the integral of IBa during the 10s prepulse normalized to cell capacitance. The solid line shows a linear fit using Deming regression. The slope of this line was not significantly different from zero (p = 0.75 for β1b expressing cells and p = 0.76 for β2a expressing cells). (C) Recovery from inactivation was voltage-dependent. Inactivation of β2a containing channels was produced by a 10s prepulse and recovery tracked at different holding potentials (-80 mV, -100 mV, or -120 mV). Solid lines show exponential fits to the mean data (-120mV holding A = 0.79, t = 24.8 s; -100mV holding A = 0.8, t = 54.7 s; -80 mV holding A = 0.62, t = 103.2 s). The bar graph plots the mean time constant calculated from fits to the individual cells (* p < 0.05, *** p < 0.001 using ANOVA and Tukey’s post-test for pairwise comparisons).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519294&req=5

pone.0134117.g002: Recovery of CaV2 channels from “slow” inactivation: dependence on voltage and CaV β subunit, but not charge density.(A) Representative IBa from HEK293 cells transfected with CaV2.2, α2δ, and either β1b or β2a subunits. In both cases inactivation was almost complete by the end of the 10s step, but the β2a subunit dramatically reduced “fast” inactivation. The right panel shows the initial ~600ms of the traces on an expanded time base. (B) Recovery rate was not correlated with barium entry. Inactivation was produced by a 10s prepulse and recovery tracked as in Fig 1F. The recovery time constant (tau) is plotted against charge density (i.e. the integral of IBa during the 10s prepulse normalized to cell capacitance. The solid line shows a linear fit using Deming regression. The slope of this line was not significantly different from zero (p = 0.75 for β1b expressing cells and p = 0.76 for β2a expressing cells). (C) Recovery from inactivation was voltage-dependent. Inactivation of β2a containing channels was produced by a 10s prepulse and recovery tracked at different holding potentials (-80 mV, -100 mV, or -120 mV). Solid lines show exponential fits to the mean data (-120mV holding A = 0.79, t = 24.8 s; -100mV holding A = 0.8, t = 54.7 s; -80 mV holding A = 0.62, t = 103.2 s). The bar graph plots the mean time constant calculated from fits to the individual cells (* p < 0.05, *** p < 0.001 using ANOVA and Tukey’s post-test for pairwise comparisons).
Mentions: Given that PMA appeared to target recovery from “slow” but not “fast” inactivation, we investigated the effects of PMA on channels containing the β2a subunit which dramatically reduces fast inactivation [20, 21]. HEK293 cells were transiently transfected with CaV2.2, α2δ, and either β2a or β1b subunits (with EGFP as a marker). As expected, inactivation of IBa during a step depolarization was much slower in β2a than β1b containing channels (Fig 2A). Recovery of β2a channels was fit with a single exponential with a time constant (at a holding potential of -100 mV) of 48.6 ± 4.7 s (n = 16), which was significantly longer compared β1b containing channels (19.5 ± 1.8 s; n = 4; p = 0.0007 unpaired t-test). As expected for voltage-dependent inactivation, the recovery kinetics of IBa showed no correlation with the amount of barium entry (Fig 2B). The overall amount of barium entry (charge density) was much greater in cells expressing the β2a subunit than in G1A1 cells (β1b expressing cells). However, when the recovery time constant was plotted against charge density, the slope of a linear fit using Deming regression was not significantly different from zero (0 = 0.75 for β1b expressing cells and p = 0.76 for β2a expressing cells). This was confirmed using Pearson’s correlation coefficient which again showed no statistically significant correlation between recovery rate and charge density: for β1b cells r = -0.13, p = 0.74; for β2a cells r = -0.08, p = 0.76. We also found that, as predicted for voltage-dependent inactivation, recovery was significantly accelerated at more hyperpolarized holding potentials (Fig 2C). Thus, recovery following the conditioning prepulse demonstrated the expected features for voltage-dependent inactivation, and the recovery kinetics were influenced by the subtype of the CaVβ subunit.

Bottom Line: The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA) had no effect.This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC.Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
CaV2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

No MeSH data available.


Related in: MedlinePlus