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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

Bisindolylmaleimide-1 and PKC(19–36) effectively block the effects of PKC / phorbol ester on Gβγ-mediated inhibition of IBa.G1A1 cells (that stably express CaV2.2, β1b and α2δ) were transiently transfected with G-protein β1 and γ2 subunits (Gβγ). (A) Representative traces showing use of a depolarizing prepulse protocol to quantify the tonic inhibition of IBa produced by Gβγ. (B) The magnitude of Gβγ-mediated inhibition was determined using the prepulse protocol (panel A) first in the absence and then in the presence of PMA (200 nM). PMA significantly diminished the Gβγ-mediated inhibition (p = 0.0015, n = 5, paired t-test). (C) Preincubation of cells with bisindolylmaleimide-1 (Bis; 500 nM) blocked the ability of PMA to reduce Gβγ-mediated inhibition (p = 0.75, n = 5, paired t-test). (C) PKC(19–36) in the patch pipette solution blocked the ability of PMA to reduce Gβγ-mediated inhibition (p = 0.09, n = 3, paired t-test).
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pone.0134117.g006: Bisindolylmaleimide-1 and PKC(19–36) effectively block the effects of PKC / phorbol ester on Gβγ-mediated inhibition of IBa.G1A1 cells (that stably express CaV2.2, β1b and α2δ) were transiently transfected with G-protein β1 and γ2 subunits (Gβγ). (A) Representative traces showing use of a depolarizing prepulse protocol to quantify the tonic inhibition of IBa produced by Gβγ. (B) The magnitude of Gβγ-mediated inhibition was determined using the prepulse protocol (panel A) first in the absence and then in the presence of PMA (200 nM). PMA significantly diminished the Gβγ-mediated inhibition (p = 0.0015, n = 5, paired t-test). (C) Preincubation of cells with bisindolylmaleimide-1 (Bis; 500 nM) blocked the ability of PMA to reduce Gβγ-mediated inhibition (p = 0.75, n = 5, paired t-test). (C) PKC(19–36) in the patch pipette solution blocked the ability of PMA to reduce Gβγ-mediated inhibition (p = 0.09, n = 3, paired t-test).

Mentions: Given the incomplete block of PMA by bisindolylmaleimide-I and especially PKC(19–36) (see above and Fig 5), we wanted to test their ability to antagonize PKC under our recording conditions. To do so, we investigated the inhibition of CaV2.2 channels by G protein βγ subunits (Gβγ) [1, 2]. Gβγ inhibits IBa by binding directly to the CaV2.2 subunit, and activation of PKC by phorbol ester has been shown to partially reverse this inhibition [38–43]. We transiently transfected G1A1 cells with GFP-tagged Gβγ subunits (Fig 6) which results in tonic inhibition of IBa. A trademark characteristic of Gβγ-mediated inhibition is that it can be reversed by strong membrane depolarization due to transient voltage-dependent dissociation of Gβγ from the channel [1]. Thus, the extent of Gβγ-mediated inhibition can be revealed using a prepulse facilitation protocol as shown in Fig 6A. Treating cells with PMA for 5-minutes significantly reduced the extent of Gβγ-mediated inhibition (Fig 6B). Pretreating cells with bisindolylmaleimide-I (Fig 6C) or PKC(19–36) (Fig 6D) blocked the ability of PMA to reduce Gβγ-mediated inhibition. Thus the drugs were effective under our recording conditions, suggesting the partial inhibition of PMA’s actions on slow inactivation is not simply due to inactive drugs or other artefacts.


"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)

Bisindolylmaleimide-1 and PKC(19–36) effectively block the effects of PKC / phorbol ester on Gβγ-mediated inhibition of IBa.G1A1 cells (that stably express CaV2.2, β1b and α2δ) were transiently transfected with G-protein β1 and γ2 subunits (Gβγ). (A) Representative traces showing use of a depolarizing prepulse protocol to quantify the tonic inhibition of IBa produced by Gβγ. (B) The magnitude of Gβγ-mediated inhibition was determined using the prepulse protocol (panel A) first in the absence and then in the presence of PMA (200 nM). PMA significantly diminished the Gβγ-mediated inhibition (p = 0.0015, n = 5, paired t-test). (C) Preincubation of cells with bisindolylmaleimide-1 (Bis; 500 nM) blocked the ability of PMA to reduce Gβγ-mediated inhibition (p = 0.75, n = 5, paired t-test). (C) PKC(19–36) in the patch pipette solution blocked the ability of PMA to reduce Gβγ-mediated inhibition (p = 0.09, n = 3, paired t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519294&req=5

pone.0134117.g006: Bisindolylmaleimide-1 and PKC(19–36) effectively block the effects of PKC / phorbol ester on Gβγ-mediated inhibition of IBa.G1A1 cells (that stably express CaV2.2, β1b and α2δ) were transiently transfected with G-protein β1 and γ2 subunits (Gβγ). (A) Representative traces showing use of a depolarizing prepulse protocol to quantify the tonic inhibition of IBa produced by Gβγ. (B) The magnitude of Gβγ-mediated inhibition was determined using the prepulse protocol (panel A) first in the absence and then in the presence of PMA (200 nM). PMA significantly diminished the Gβγ-mediated inhibition (p = 0.0015, n = 5, paired t-test). (C) Preincubation of cells with bisindolylmaleimide-1 (Bis; 500 nM) blocked the ability of PMA to reduce Gβγ-mediated inhibition (p = 0.75, n = 5, paired t-test). (C) PKC(19–36) in the patch pipette solution blocked the ability of PMA to reduce Gβγ-mediated inhibition (p = 0.09, n = 3, paired t-test).
Mentions: Given the incomplete block of PMA by bisindolylmaleimide-I and especially PKC(19–36) (see above and Fig 5), we wanted to test their ability to antagonize PKC under our recording conditions. To do so, we investigated the inhibition of CaV2.2 channels by G protein βγ subunits (Gβγ) [1, 2]. Gβγ inhibits IBa by binding directly to the CaV2.2 subunit, and activation of PKC by phorbol ester has been shown to partially reverse this inhibition [38–43]. We transiently transfected G1A1 cells with GFP-tagged Gβγ subunits (Fig 6) which results in tonic inhibition of IBa. A trademark characteristic of Gβγ-mediated inhibition is that it can be reversed by strong membrane depolarization due to transient voltage-dependent dissociation of Gβγ from the channel [1]. Thus, the extent of Gβγ-mediated inhibition can be revealed using a prepulse facilitation protocol as shown in Fig 6A. Treating cells with PMA for 5-minutes significantly reduced the extent of Gβγ-mediated inhibition (Fig 6B). Pretreating cells with bisindolylmaleimide-I (Fig 6C) or PKC(19–36) (Fig 6D) blocked the ability of PMA to reduce Gβγ-mediated inhibition. Thus the drugs were effective under our recording conditions, suggesting the partial inhibition of PMA’s actions on slow inactivation is not simply due to inactive drugs or other artefacts.

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