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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

Intracellular application of GDP-β-S or GTP-γ-S reduced the effect of PMA on recovery from inactivation.(A) HEK293 cells expressing CaV2.2, β1b and α2δ were recorded with patch pipette solution containing 0.5mM GDP-β-S and stimulated with a 5Hz/10s train. Fractional recovery from inactivation is plotted over time and the solid lines show a double exponential fit to the mean data (fit parameters: GDP-β-S A1 = 0.13, A2 = 0.71, t1 = 0.9 s, t2 = 35.0 s; GDP-β-S + PMA A1 = 0.18, A2 = 0.61, t1 = 3.6 s, t2 = 62.7 s, comparison of fits F = 12.9 p < 0.0001). (B) The change in recovery rate produced by PMA (tau in the presence of PMA / tau before application of PMA) for control cells, cells recorded with GDP-β-S in the patch pipette solution, and cells recorded with GTP-γ-S in the patch pipette solution (ns, not significant, * p < 0.05, ** p < 0.01; ANOVA with Bonferroni’s post-test for multiple pairwise comparisons). (C) Endogenous ICa was recorded from adrenal chromaffin cells with GDP-β-S in the patch pipette solution. Chromaffin cells were stimulated with a 10s step depolarization and recovery from inactivation was tracked at the indicated time points before and during application of PMA. The mean recovery data was fit with a double exponential (fit parameters: GDP-β-S Y0 = 0.02, A1 = 0.36, A2 = 0.52, t1 = 0.8 s, t2 = 28.1 s; GDP-β-S + PMA Y0 = 0.02, A1 = 0.25, A2 = 0.66, t1 = 0.8 s, t2 = 37.4 s, comparison of fits F = 1.65 p = 0.2). (D) Mean (± sem) data are shown for the slower of the two time constants from control cells (Ctl, n = 7) and cells recorded with GDP-β-S (n = 4). GDP-β-S had no effect on recovery rate in the absence of PMA (black bars) but significantly reduced the slowing of recovery produced by PMA (red bars) (ns, not significant, * p < 0.05; ANOVA with Bonferroni’s post-test for multiple pairwise comparisons).
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pone.0134117.g008: Intracellular application of GDP-β-S or GTP-γ-S reduced the effect of PMA on recovery from inactivation.(A) HEK293 cells expressing CaV2.2, β1b and α2δ were recorded with patch pipette solution containing 0.5mM GDP-β-S and stimulated with a 5Hz/10s train. Fractional recovery from inactivation is plotted over time and the solid lines show a double exponential fit to the mean data (fit parameters: GDP-β-S A1 = 0.13, A2 = 0.71, t1 = 0.9 s, t2 = 35.0 s; GDP-β-S + PMA A1 = 0.18, A2 = 0.61, t1 = 3.6 s, t2 = 62.7 s, comparison of fits F = 12.9 p < 0.0001). (B) The change in recovery rate produced by PMA (tau in the presence of PMA / tau before application of PMA) for control cells, cells recorded with GDP-β-S in the patch pipette solution, and cells recorded with GTP-γ-S in the patch pipette solution (ns, not significant, * p < 0.05, ** p < 0.01; ANOVA with Bonferroni’s post-test for multiple pairwise comparisons). (C) Endogenous ICa was recorded from adrenal chromaffin cells with GDP-β-S in the patch pipette solution. Chromaffin cells were stimulated with a 10s step depolarization and recovery from inactivation was tracked at the indicated time points before and during application of PMA. The mean recovery data was fit with a double exponential (fit parameters: GDP-β-S Y0 = 0.02, A1 = 0.36, A2 = 0.52, t1 = 0.8 s, t2 = 28.1 s; GDP-β-S + PMA Y0 = 0.02, A1 = 0.25, A2 = 0.66, t1 = 0.8 s, t2 = 37.4 s, comparison of fits F = 1.65 p = 0.2). (D) Mean (± sem) data are shown for the slower of the two time constants from control cells (Ctl, n = 7) and cells recorded with GDP-β-S (n = 4). GDP-β-S had no effect on recovery rate in the absence of PMA (black bars) but significantly reduced the slowing of recovery produced by PMA (red bars) (ns, not significant, * p < 0.05; ANOVA with Bonferroni’s post-test for multiple pairwise comparisons).

Mentions: To investigate any potential involvement of G protein signaling in the effects of PMA we replaced GTP with GDP-β-S (0.5 mM) in the intracellular patch pipette solution to prevent activation of heterotrimeric and monomeric G proteins (Fig 8). First we used this solution to record from HEK cells expressing CaV2.2, β1b and α2δ subunits stimulated with a 5Hz/10s train. In the absence of PMA, GDP-β-S increased the extent of inactivation at the end of the 5Hz train compared to control cells (50 ± 4%, n = 8 Vs 34 ± 2%, n = 6; p = 0.011, unpaired t-test). The rate of recovery from inactivation under control conditions (before application of PMA) was not altered by GDP-β-S, but the slowing of recovery produced by PMA was significantly reduced, as reflected in the tau ratio (Fig 8A and 8B). We repeated a similar experiment but with GTP-γ-S added to the patch-pipette solution. Again, the prolonged recovery from inactivation produced by PMA was blocked in these cells (Fig 8B). Similarly, in adrenal chromaffin cells GDP-β-S had no effect on recovery from inactivation under control conditions, but significantly reduced the slowing of recovery produced by PMA (Fig 8C and 8D). Together these data suggest that G protein signaling pathways can influence the kinetics of recovery from inactivation, opposing the actions of PMA.


"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)

Intracellular application of GDP-β-S or GTP-γ-S reduced the effect of PMA on recovery from inactivation.(A) HEK293 cells expressing CaV2.2, β1b and α2δ were recorded with patch pipette solution containing 0.5mM GDP-β-S and stimulated with a 5Hz/10s train. Fractional recovery from inactivation is plotted over time and the solid lines show a double exponential fit to the mean data (fit parameters: GDP-β-S A1 = 0.13, A2 = 0.71, t1 = 0.9 s, t2 = 35.0 s; GDP-β-S + PMA A1 = 0.18, A2 = 0.61, t1 = 3.6 s, t2 = 62.7 s, comparison of fits F = 12.9 p < 0.0001). (B) The change in recovery rate produced by PMA (tau in the presence of PMA / tau before application of PMA) for control cells, cells recorded with GDP-β-S in the patch pipette solution, and cells recorded with GTP-γ-S in the patch pipette solution (ns, not significant, * p < 0.05, ** p < 0.01; ANOVA with Bonferroni’s post-test for multiple pairwise comparisons). (C) Endogenous ICa was recorded from adrenal chromaffin cells with GDP-β-S in the patch pipette solution. Chromaffin cells were stimulated with a 10s step depolarization and recovery from inactivation was tracked at the indicated time points before and during application of PMA. The mean recovery data was fit with a double exponential (fit parameters: GDP-β-S Y0 = 0.02, A1 = 0.36, A2 = 0.52, t1 = 0.8 s, t2 = 28.1 s; GDP-β-S + PMA Y0 = 0.02, A1 = 0.25, A2 = 0.66, t1 = 0.8 s, t2 = 37.4 s, comparison of fits F = 1.65 p = 0.2). (D) Mean (± sem) data are shown for the slower of the two time constants from control cells (Ctl, n = 7) and cells recorded with GDP-β-S (n = 4). GDP-β-S had no effect on recovery rate in the absence of PMA (black bars) but significantly reduced the slowing of recovery produced by PMA (red bars) (ns, not significant, * p < 0.05; ANOVA with Bonferroni’s post-test for multiple pairwise comparisons).
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Related In: Results  -  Collection

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pone.0134117.g008: Intracellular application of GDP-β-S or GTP-γ-S reduced the effect of PMA on recovery from inactivation.(A) HEK293 cells expressing CaV2.2, β1b and α2δ were recorded with patch pipette solution containing 0.5mM GDP-β-S and stimulated with a 5Hz/10s train. Fractional recovery from inactivation is plotted over time and the solid lines show a double exponential fit to the mean data (fit parameters: GDP-β-S A1 = 0.13, A2 = 0.71, t1 = 0.9 s, t2 = 35.0 s; GDP-β-S + PMA A1 = 0.18, A2 = 0.61, t1 = 3.6 s, t2 = 62.7 s, comparison of fits F = 12.9 p < 0.0001). (B) The change in recovery rate produced by PMA (tau in the presence of PMA / tau before application of PMA) for control cells, cells recorded with GDP-β-S in the patch pipette solution, and cells recorded with GTP-γ-S in the patch pipette solution (ns, not significant, * p < 0.05, ** p < 0.01; ANOVA with Bonferroni’s post-test for multiple pairwise comparisons). (C) Endogenous ICa was recorded from adrenal chromaffin cells with GDP-β-S in the patch pipette solution. Chromaffin cells were stimulated with a 10s step depolarization and recovery from inactivation was tracked at the indicated time points before and during application of PMA. The mean recovery data was fit with a double exponential (fit parameters: GDP-β-S Y0 = 0.02, A1 = 0.36, A2 = 0.52, t1 = 0.8 s, t2 = 28.1 s; GDP-β-S + PMA Y0 = 0.02, A1 = 0.25, A2 = 0.66, t1 = 0.8 s, t2 = 37.4 s, comparison of fits F = 1.65 p = 0.2). (D) Mean (± sem) data are shown for the slower of the two time constants from control cells (Ctl, n = 7) and cells recorded with GDP-β-S (n = 4). GDP-β-S had no effect on recovery rate in the absence of PMA (black bars) but significantly reduced the slowing of recovery produced by PMA (red bars) (ns, not significant, * p < 0.05; ANOVA with Bonferroni’s post-test for multiple pairwise comparisons).
Mentions: To investigate any potential involvement of G protein signaling in the effects of PMA we replaced GTP with GDP-β-S (0.5 mM) in the intracellular patch pipette solution to prevent activation of heterotrimeric and monomeric G proteins (Fig 8). First we used this solution to record from HEK cells expressing CaV2.2, β1b and α2δ subunits stimulated with a 5Hz/10s train. In the absence of PMA, GDP-β-S increased the extent of inactivation at the end of the 5Hz train compared to control cells (50 ± 4%, n = 8 Vs 34 ± 2%, n = 6; p = 0.011, unpaired t-test). The rate of recovery from inactivation under control conditions (before application of PMA) was not altered by GDP-β-S, but the slowing of recovery produced by PMA was significantly reduced, as reflected in the tau ratio (Fig 8A and 8B). We repeated a similar experiment but with GTP-γ-S added to the patch-pipette solution. Again, the prolonged recovery from inactivation produced by PMA was blocked in these cells (Fig 8B). Similarly, in adrenal chromaffin cells GDP-β-S had no effect on recovery from inactivation under control conditions, but significantly reduced the slowing of recovery produced by PMA (Fig 8C and 8D). Together these data suggest that G protein signaling pathways can influence the kinetics of recovery from inactivation, opposing the actions of PMA.

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