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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

PMA significantly prolonged recovery from inactivation in β2a containing channels.(A) HEK293 cells were transfected with CaV2.2, α2δ, and β2a subunits. Inactivation was produced by a 10s prepulse and recovery tracked as in Fig 1F. This was done twice in each cell: in some cells PMA was applied 5-min before the second stimulation protocol (left panel), while other cells were used as time-matched controls (i.e. no PMA was applied; right panel). Solid lines show exponential fits to the mean data (left panel: control A = 0.82, t = 40.4 s; PMA A = 0.65, t = 101 s, comparison of fits F = 24.2 p < 0.0001: right panel control A = 0.74, t = 36.9 s; control repeat A = 0.78, t = 55.8 s, comparison of fits F = 2.28 p = 0.1). (B) The time constant of the exponential fit to each individual cell was calculated and the means plotted. PMA significantly prolonged recovery from inactivation (* p < 0.05, ** p < 0.01, ns = “not significant” using ANOVA followed by Bonferroni’s multiple comparison test).
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pone.0134117.g003: PMA significantly prolonged recovery from inactivation in β2a containing channels.(A) HEK293 cells were transfected with CaV2.2, α2δ, and β2a subunits. Inactivation was produced by a 10s prepulse and recovery tracked as in Fig 1F. This was done twice in each cell: in some cells PMA was applied 5-min before the second stimulation protocol (left panel), while other cells were used as time-matched controls (i.e. no PMA was applied; right panel). Solid lines show exponential fits to the mean data (left panel: control A = 0.82, t = 40.4 s; PMA A = 0.65, t = 101 s, comparison of fits F = 24.2 p < 0.0001: right panel control A = 0.74, t = 36.9 s; control repeat A = 0.78, t = 55.8 s, comparison of fits F = 2.28 p = 0.1). (B) The time constant of the exponential fit to each individual cell was calculated and the means plotted. PMA significantly prolonged recovery from inactivation (* p < 0.05, ** p < 0.01, ns = “not significant” using ANOVA followed by Bonferroni’s multiple comparison test).

Mentions: PMA significantly prolonged the recovery time constant in β2a containing channels from 46.7 ± 9.4 s to 108.6 ± 9.9 s (n = 6, p = 0.00001, paired t-test) (Fig 3). We also tested the closely related CaV2.1 (P/Q-type) channel (CaV2.1, β2a and α2δ). Recovery from inactivation in CaV2.1 channels was faster than in CaV2.2 channels, but was still significantly prolonged by PMA from 14.8 ± 3.2 s to 35.5 ± 9.7 s (n = 5, p = 0.04, paired t-test). To compare the effects of PMA across these channels with different subunit combinations that had inherently different rates of recovery, we calculated the change in recovery time constant (i.e. tau in the presence of PMA / tau before application of PMA). This tau ratio showed that PMA prolonged recovery from inactivation to a similar extent in all cases: the tau ratio was 2.66 ± 0.35 for CaV2.2 + β2a (n = 6), 3.09 ± 0.3 for CaV2.2 + β1b (n = 9), and 2.38 ± 0.38 for CaV2.1 + β2a (n = 5) (p = 0.34; F = 1.15, One-way ANOVA).


"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)

PMA significantly prolonged recovery from inactivation in β2a containing channels.(A) HEK293 cells were transfected with CaV2.2, α2δ, and β2a subunits. Inactivation was produced by a 10s prepulse and recovery tracked as in Fig 1F. This was done twice in each cell: in some cells PMA was applied 5-min before the second stimulation protocol (left panel), while other cells were used as time-matched controls (i.e. no PMA was applied; right panel). Solid lines show exponential fits to the mean data (left panel: control A = 0.82, t = 40.4 s; PMA A = 0.65, t = 101 s, comparison of fits F = 24.2 p < 0.0001: right panel control A = 0.74, t = 36.9 s; control repeat A = 0.78, t = 55.8 s, comparison of fits F = 2.28 p = 0.1). (B) The time constant of the exponential fit to each individual cell was calculated and the means plotted. PMA significantly prolonged recovery from inactivation (* p < 0.05, ** p < 0.01, ns = “not significant” using ANOVA followed by Bonferroni’s multiple comparison test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519294&req=5

pone.0134117.g003: PMA significantly prolonged recovery from inactivation in β2a containing channels.(A) HEK293 cells were transfected with CaV2.2, α2δ, and β2a subunits. Inactivation was produced by a 10s prepulse and recovery tracked as in Fig 1F. This was done twice in each cell: in some cells PMA was applied 5-min before the second stimulation protocol (left panel), while other cells were used as time-matched controls (i.e. no PMA was applied; right panel). Solid lines show exponential fits to the mean data (left panel: control A = 0.82, t = 40.4 s; PMA A = 0.65, t = 101 s, comparison of fits F = 24.2 p < 0.0001: right panel control A = 0.74, t = 36.9 s; control repeat A = 0.78, t = 55.8 s, comparison of fits F = 2.28 p = 0.1). (B) The time constant of the exponential fit to each individual cell was calculated and the means plotted. PMA significantly prolonged recovery from inactivation (* p < 0.05, ** p < 0.01, ns = “not significant” using ANOVA followed by Bonferroni’s multiple comparison test).
Mentions: PMA significantly prolonged the recovery time constant in β2a containing channels from 46.7 ± 9.4 s to 108.6 ± 9.9 s (n = 6, p = 0.00001, paired t-test) (Fig 3). We also tested the closely related CaV2.1 (P/Q-type) channel (CaV2.1, β2a and α2δ). Recovery from inactivation in CaV2.1 channels was faster than in CaV2.2 channels, but was still significantly prolonged by PMA from 14.8 ± 3.2 s to 35.5 ± 9.7 s (n = 5, p = 0.04, paired t-test). To compare the effects of PMA across these channels with different subunit combinations that had inherently different rates of recovery, we calculated the change in recovery time constant (i.e. tau in the presence of PMA / tau before application of PMA). This tau ratio showed that PMA prolonged recovery from inactivation to a similar extent in all cases: the tau ratio was 2.66 ± 0.35 for CaV2.2 + β2a (n = 6), 3.09 ± 0.3 for CaV2.2 + β1b (n = 9), and 2.38 ± 0.38 for CaV2.1 + β2a (n = 5) (p = 0.34; F = 1.15, One-way ANOVA).

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