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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 from “slow” inactivation following trains of brief stimuli was prolonged by PMA.(A) HEK293 cells expressing CaV2.2, β1b and α2δ were stimulated with a train of brief (8ms) step depolarizations (50Hz for 1s) and recovery from inactivation was tracked at various time points as indicated. IBa amplitude was normalized to the first pulse of the stimulus train. The left panel shows the current decline (inactivation) during the stimulus train and the right panel shows the time-course of recovery following the train (note the change in scale on the X axis). Solid lines show double exponential fits to the mean recovery data (control: Y0 = 0.27, A1 = 0.39, A2 = 0.27, t1 = 0.38 s, t2 = 24.8 s: PMA Y0 = 0.31, A1 = 0.33, A2 = 0.16, t1 = 0.56 s, t2 = 68.8 s, comparison of fits F = 8.98 p = 0.001). The inset bar graph shows the mean time constant for the slower component of recovery calculated from fits to the individual cells (* p = 0.045 paired t-test, n = 4). (B) The same layout as panel A, except that the cells were stimulated using a 5Hz train lasting 10 s. Solid lines show an exponential fit to the mean recovery data (control: Y0 = 0.67, A = 0.30, t = 32.4 s; PMA Y0 = 0.60, A = 0.31, t = 106.8 s, comparison of fits F = 79.25 p < 0.0001). The inset bar graph shows the mean time constant for recovery calculated from fits to the individual cells (** p = 0.0042 paired t-test, n = 6).
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pone.0134117.g004: Recovery from “slow” inactivation following trains of brief stimuli was prolonged by PMA.(A) HEK293 cells expressing CaV2.2, β1b and α2δ were stimulated with a train of brief (8ms) step depolarizations (50Hz for 1s) and recovery from inactivation was tracked at various time points as indicated. IBa amplitude was normalized to the first pulse of the stimulus train. The left panel shows the current decline (inactivation) during the stimulus train and the right panel shows the time-course of recovery following the train (note the change in scale on the X axis). Solid lines show double exponential fits to the mean recovery data (control: Y0 = 0.27, A1 = 0.39, A2 = 0.27, t1 = 0.38 s, t2 = 24.8 s: PMA Y0 = 0.31, A1 = 0.33, A2 = 0.16, t1 = 0.56 s, t2 = 68.8 s, comparison of fits F = 8.98 p = 0.001). The inset bar graph shows the mean time constant for the slower component of recovery calculated from fits to the individual cells (* p = 0.045 paired t-test, n = 4). (B) The same layout as panel A, except that the cells were stimulated using a 5Hz train lasting 10 s. Solid lines show an exponential fit to the mean recovery data (control: Y0 = 0.67, A = 0.30, t = 32.4 s; PMA Y0 = 0.60, A = 0.31, t = 106.8 s, comparison of fits F = 79.25 p < 0.0001). The inset bar graph shows the mean time constant for recovery calculated from fits to the individual cells (** p = 0.0042 paired t-test, n = 6).

Mentions: To more closely mimic physiologically relevant electrical activity, we stimulated cells with trains of brief depolarizations applied at 50Hz for 1s, or 5Hz for 10s (Fig 4). The extent of inactivation at the end of the 50Hz train was not altered by PMA (73% ± 3.6% in control conditions Vs. 69 ± 3.6% in PMA; n = 4; p = 0.21, paired t test) (Fig 4A). The recovery was biphasic, and PMA slightly reduced the magnitude of the initial fast component: in control conditions 52 ± 3% of the inactivation was recovered by 1s (the first recovery time point) and this was reduced to 40 ± 4% by PMA (n = 4, p = 0.021, paired t-test). PMA significantly prolonged the time constant of the slow recovery phase from 29.7 ± 4.6 s in control conditions to 112.9 ± 23.9 s in the presence of PMA (n = 4; p = 0.045 paired t-test). As expected, the 5Hz train produced less inactivation than the 50Hz train (Fig 4B). The extent of inactivation at the end of the 5Hz train was significantly increased by PMA (33 ± 2% Vs. 41 ± 3%, n = 6; p = 0.015, paired t-test). Recovery following the 5 Hz train was well fit with a single exponential and lacked the initial fast component seen after the 50 Hz train. The time constant of recovery was significantly slowed from 35.1 ± 6.7 s in control conditions to 118.5 ± 13.8 s in the presence of PMA (n = 6, p = 0.0042, paired t-test). These data show that PMA prolonged recovery from “slow” inactivation following physiologically relevant stimulus trains as well as following sustained step depolarization.


"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 from “slow” inactivation following trains of brief stimuli was prolonged by PMA.(A) HEK293 cells expressing CaV2.2, β1b and α2δ were stimulated with a train of brief (8ms) step depolarizations (50Hz for 1s) and recovery from inactivation was tracked at various time points as indicated. IBa amplitude was normalized to the first pulse of the stimulus train. The left panel shows the current decline (inactivation) during the stimulus train and the right panel shows the time-course of recovery following the train (note the change in scale on the X axis). Solid lines show double exponential fits to the mean recovery data (control: Y0 = 0.27, A1 = 0.39, A2 = 0.27, t1 = 0.38 s, t2 = 24.8 s: PMA Y0 = 0.31, A1 = 0.33, A2 = 0.16, t1 = 0.56 s, t2 = 68.8 s, comparison of fits F = 8.98 p = 0.001). The inset bar graph shows the mean time constant for the slower component of recovery calculated from fits to the individual cells (* p = 0.045 paired t-test, n = 4). (B) The same layout as panel A, except that the cells were stimulated using a 5Hz train lasting 10 s. Solid lines show an exponential fit to the mean recovery data (control: Y0 = 0.67, A = 0.30, t = 32.4 s; PMA Y0 = 0.60, A = 0.31, t = 106.8 s, comparison of fits F = 79.25 p < 0.0001). The inset bar graph shows the mean time constant for recovery calculated from fits to the individual cells (** p = 0.0042 paired t-test, n = 6).
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Related In: Results  -  Collection

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pone.0134117.g004: Recovery from “slow” inactivation following trains of brief stimuli was prolonged by PMA.(A) HEK293 cells expressing CaV2.2, β1b and α2δ were stimulated with a train of brief (8ms) step depolarizations (50Hz for 1s) and recovery from inactivation was tracked at various time points as indicated. IBa amplitude was normalized to the first pulse of the stimulus train. The left panel shows the current decline (inactivation) during the stimulus train and the right panel shows the time-course of recovery following the train (note the change in scale on the X axis). Solid lines show double exponential fits to the mean recovery data (control: Y0 = 0.27, A1 = 0.39, A2 = 0.27, t1 = 0.38 s, t2 = 24.8 s: PMA Y0 = 0.31, A1 = 0.33, A2 = 0.16, t1 = 0.56 s, t2 = 68.8 s, comparison of fits F = 8.98 p = 0.001). The inset bar graph shows the mean time constant for the slower component of recovery calculated from fits to the individual cells (* p = 0.045 paired t-test, n = 4). (B) The same layout as panel A, except that the cells were stimulated using a 5Hz train lasting 10 s. Solid lines show an exponential fit to the mean recovery data (control: Y0 = 0.67, A = 0.30, t = 32.4 s; PMA Y0 = 0.60, A = 0.31, t = 106.8 s, comparison of fits F = 79.25 p < 0.0001). The inset bar graph shows the mean time constant for recovery calculated from fits to the individual cells (** p = 0.0042 paired t-test, n = 6).
Mentions: To more closely mimic physiologically relevant electrical activity, we stimulated cells with trains of brief depolarizations applied at 50Hz for 1s, or 5Hz for 10s (Fig 4). The extent of inactivation at the end of the 50Hz train was not altered by PMA (73% ± 3.6% in control conditions Vs. 69 ± 3.6% in PMA; n = 4; p = 0.21, paired t test) (Fig 4A). The recovery was biphasic, and PMA slightly reduced the magnitude of the initial fast component: in control conditions 52 ± 3% of the inactivation was recovered by 1s (the first recovery time point) and this was reduced to 40 ± 4% by PMA (n = 4, p = 0.021, paired t-test). PMA significantly prolonged the time constant of the slow recovery phase from 29.7 ± 4.6 s in control conditions to 112.9 ± 23.9 s in the presence of PMA (n = 4; p = 0.045 paired t-test). As expected, the 5Hz train produced less inactivation than the 50Hz train (Fig 4B). The extent of inactivation at the end of the 5Hz train was significantly increased by PMA (33 ± 2% Vs. 41 ± 3%, n = 6; p = 0.015, paired t-test). Recovery following the 5 Hz train was well fit with a single exponential and lacked the initial fast component seen after the 50 Hz train. The time constant of recovery was significantly slowed from 35.1 ± 6.7 s in control conditions to 118.5 ± 13.8 s in the presence of PMA (n = 6, p = 0.0042, paired t-test). These data show that PMA prolonged recovery from “slow” inactivation following physiologically relevant stimulus trains as well as following sustained step depolarization.

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