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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

Phorbol ester (PMA) selectively targets recovery from “slow” inactivation.The effects of acute application of PMA on HEK293 cells expressing CaV2.2, β1b and α2δ were investigated. (A) Cells were stimulated with a 10ms step depolarization and peak amplitude of IBa plotted against time (normalized to the time point immediately before PMA application) (n = 8; error bars are plotted but in most cases fall within the symbol so are not visible). The inset bar graph shows the change in IBa amplitude after 5-minutes of PMA (200 nM) was not different from time-matched control cells. (B) Shows the normalized current-voltage relationship of IBa evoked by a ramp depolarization first in the absence (Ctl) then in the presence of PMA (200 nM for 5-min). Traces are the mean values (n = 7) and error bars are omitted for clarity. (C) The voltage-dependence of closed-state inactivation was determined before and during application of PMA (protocol shown in the inset). The mean normalized current amplitude was plotted and fit with a Boltzmann function of the form: I = I2 + (I1 —I2) / 1 + e((V—V50)/k)). The two curves (control and PMA) were not significantly different from one another (F = 0.97 p = 0.45); V50 = -49 mV in control and -51 mV in PMA, slope = -7.38 in control and -7.4 in PMA. (D) Inactivation of IBa during a 1s step depolarization was not altered by PMA. Control cells or cells treated with PMA for 5–10 minutes were stimulated with a 1s step to +10mV and the evoked currents normalized to the peak amplitude to enable better comparison of the inactivation time-course. Traces show the means but error bars are omitted for clarity. (E) Recovery from “fast” inactivation was not significantly different in control cells or PMA treated cells (200nM for 5–10 minutes). Inactivation of IBa was produced by 1s prepulse and recovery determined by a brief test pulse after the indicated interval. This was repeated once every 60s (see inset above graph for voltage-protocol). Current amplitude during the recovery test pulse was normalized to peak IBa evoked by the prepulse. The solid lines show double exponential fits to the data. (F) PMA prolonged recovery from “slow inactivation”. Inactivation of IBa was produced by 10s prepulse and recovery was tracked using a series of brief test pulses applied at the indicated time points following the 10s prepulse (see inset). This was repeated twice in the same cell, once before application of PMA ((Ctl) and once in the presence of PMA (after 5-min exposure). Solid lines show double exponential fits to the mean data (control A1 = 0.23, A2 = 0.73, t1 = 1.21 s, t2 = 18.96 s; PMA A1 = 0.12, A2 = 0.76, t1 = 1.06 s, t2 = 49.2 s, fit comparison F = 27.6 p < 0.0001). The inset bar graph shows the mean time-constant for the slow phase of recovery calculated from fits to the individual cells (*** p = 0.001, paired t-test).
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pone.0134117.g001: Phorbol ester (PMA) selectively targets recovery from “slow” inactivation.The effects of acute application of PMA on HEK293 cells expressing CaV2.2, β1b and α2δ were investigated. (A) Cells were stimulated with a 10ms step depolarization and peak amplitude of IBa plotted against time (normalized to the time point immediately before PMA application) (n = 8; error bars are plotted but in most cases fall within the symbol so are not visible). The inset bar graph shows the change in IBa amplitude after 5-minutes of PMA (200 nM) was not different from time-matched control cells. (B) Shows the normalized current-voltage relationship of IBa evoked by a ramp depolarization first in the absence (Ctl) then in the presence of PMA (200 nM for 5-min). Traces are the mean values (n = 7) and error bars are omitted for clarity. (C) The voltage-dependence of closed-state inactivation was determined before and during application of PMA (protocol shown in the inset). The mean normalized current amplitude was plotted and fit with a Boltzmann function of the form: I = I2 + (I1 —I2) / 1 + e((V—V50)/k)). The two curves (control and PMA) were not significantly different from one another (F = 0.97 p = 0.45); V50 = -49 mV in control and -51 mV in PMA, slope = -7.38 in control and -7.4 in PMA. (D) Inactivation of IBa during a 1s step depolarization was not altered by PMA. Control cells or cells treated with PMA for 5–10 minutes were stimulated with a 1s step to +10mV and the evoked currents normalized to the peak amplitude to enable better comparison of the inactivation time-course. Traces show the means but error bars are omitted for clarity. (E) Recovery from “fast” inactivation was not significantly different in control cells or PMA treated cells (200nM for 5–10 minutes). Inactivation of IBa was produced by 1s prepulse and recovery determined by a brief test pulse after the indicated interval. This was repeated once every 60s (see inset above graph for voltage-protocol). Current amplitude during the recovery test pulse was normalized to peak IBa evoked by the prepulse. The solid lines show double exponential fits to the data. (F) PMA prolonged recovery from “slow inactivation”. Inactivation of IBa was produced by 10s prepulse and recovery was tracked using a series of brief test pulses applied at the indicated time points following the 10s prepulse (see inset). This was repeated twice in the same cell, once before application of PMA ((Ctl) and once in the presence of PMA (after 5-min exposure). Solid lines show double exponential fits to the mean data (control A1 = 0.23, A2 = 0.73, t1 = 1.21 s, t2 = 18.96 s; PMA A1 = 0.12, A2 = 0.76, t1 = 1.06 s, t2 = 49.2 s, fit comparison F = 27.6 p < 0.0001). The inset bar graph shows the mean time-constant for the slow phase of recovery calculated from fits to the individual cells (*** p = 0.001, paired t-test).

Mentions: We set out to investigate voltage-dependent inactivation of CaV2.2 Ca2+ channels and how it might be regulated by the PKC-activator PMA. To isolate voltage-dependent inactivation, we used barium rather than calcium as the extracellular divalent cation and included BAPTA (10 mM) in the intracellular patch-pipette solution to prevent calcium-dependent inactivation. G1A1 cells stably express CaV2.2, α2δ and β1b calcium channel subunits. Under our recording conditions, acute application of PMA (200 nM for 5-minutes) had no effect on the peak barium current (IBa) amplitude (Fig 1), and did not shift the current-voltage-relationship (Fig 1B), the voltage-dependence of inactivation (Fig 1C), or the rate/extent of inactivation during a 1s step depolarization (Fig 1D). To assess recovery of IBa from “fast” inactivation, we used a standard protocol in which a 1s prepulse (used to produce inactivation) was followed by a short test pulse to determine the extent of recovery (Fig 1E). This double pulse protocol was repeated every 60s and the recovery interval following the prepulse increased with each stimulus. Recovery following the 1s step was relatively fast (~ 50% recovery within 1s). Due to concerns with rundown and the possibility of a much slower component to the recovery, we limited this protocol to five repeats, covering the first 6-seconds of recovery. These data were fit well with a double exponential (Fig 1E) and neither of the fitted time constants were significantly changed in PMA treated cells (tau fast = 167 ± 12 ms in controls Vs. 161 ± 6 ms in PMA treated cells, p = 0.67 unpaired t-test: tau slow = 2.46 ± 0.49 s in controls Vs 3.1 ± 0.21 s in PMA treated cells, p = 0.23 unpaired t test).


"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)

Phorbol ester (PMA) selectively targets recovery from “slow” inactivation.The effects of acute application of PMA on HEK293 cells expressing CaV2.2, β1b and α2δ were investigated. (A) Cells were stimulated with a 10ms step depolarization and peak amplitude of IBa plotted against time (normalized to the time point immediately before PMA application) (n = 8; error bars are plotted but in most cases fall within the symbol so are not visible). The inset bar graph shows the change in IBa amplitude after 5-minutes of PMA (200 nM) was not different from time-matched control cells. (B) Shows the normalized current-voltage relationship of IBa evoked by a ramp depolarization first in the absence (Ctl) then in the presence of PMA (200 nM for 5-min). Traces are the mean values (n = 7) and error bars are omitted for clarity. (C) The voltage-dependence of closed-state inactivation was determined before and during application of PMA (protocol shown in the inset). The mean normalized current amplitude was plotted and fit with a Boltzmann function of the form: I = I2 + (I1 —I2) / 1 + e((V—V50)/k)). The two curves (control and PMA) were not significantly different from one another (F = 0.97 p = 0.45); V50 = -49 mV in control and -51 mV in PMA, slope = -7.38 in control and -7.4 in PMA. (D) Inactivation of IBa during a 1s step depolarization was not altered by PMA. Control cells or cells treated with PMA for 5–10 minutes were stimulated with a 1s step to +10mV and the evoked currents normalized to the peak amplitude to enable better comparison of the inactivation time-course. Traces show the means but error bars are omitted for clarity. (E) Recovery from “fast” inactivation was not significantly different in control cells or PMA treated cells (200nM for 5–10 minutes). Inactivation of IBa was produced by 1s prepulse and recovery determined by a brief test pulse after the indicated interval. This was repeated once every 60s (see inset above graph for voltage-protocol). Current amplitude during the recovery test pulse was normalized to peak IBa evoked by the prepulse. The solid lines show double exponential fits to the data. (F) PMA prolonged recovery from “slow inactivation”. Inactivation of IBa was produced by 10s prepulse and recovery was tracked using a series of brief test pulses applied at the indicated time points following the 10s prepulse (see inset). This was repeated twice in the same cell, once before application of PMA ((Ctl) and once in the presence of PMA (after 5-min exposure). Solid lines show double exponential fits to the mean data (control A1 = 0.23, A2 = 0.73, t1 = 1.21 s, t2 = 18.96 s; PMA A1 = 0.12, A2 = 0.76, t1 = 1.06 s, t2 = 49.2 s, fit comparison F = 27.6 p < 0.0001). The inset bar graph shows the mean time-constant for the slow phase of recovery calculated from fits to the individual cells (*** p = 0.001, paired t-test).
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pone.0134117.g001: Phorbol ester (PMA) selectively targets recovery from “slow” inactivation.The effects of acute application of PMA on HEK293 cells expressing CaV2.2, β1b and α2δ were investigated. (A) Cells were stimulated with a 10ms step depolarization and peak amplitude of IBa plotted against time (normalized to the time point immediately before PMA application) (n = 8; error bars are plotted but in most cases fall within the symbol so are not visible). The inset bar graph shows the change in IBa amplitude after 5-minutes of PMA (200 nM) was not different from time-matched control cells. (B) Shows the normalized current-voltage relationship of IBa evoked by a ramp depolarization first in the absence (Ctl) then in the presence of PMA (200 nM for 5-min). Traces are the mean values (n = 7) and error bars are omitted for clarity. (C) The voltage-dependence of closed-state inactivation was determined before and during application of PMA (protocol shown in the inset). The mean normalized current amplitude was plotted and fit with a Boltzmann function of the form: I = I2 + (I1 —I2) / 1 + e((V—V50)/k)). The two curves (control and PMA) were not significantly different from one another (F = 0.97 p = 0.45); V50 = -49 mV in control and -51 mV in PMA, slope = -7.38 in control and -7.4 in PMA. (D) Inactivation of IBa during a 1s step depolarization was not altered by PMA. Control cells or cells treated with PMA for 5–10 minutes were stimulated with a 1s step to +10mV and the evoked currents normalized to the peak amplitude to enable better comparison of the inactivation time-course. Traces show the means but error bars are omitted for clarity. (E) Recovery from “fast” inactivation was not significantly different in control cells or PMA treated cells (200nM for 5–10 minutes). Inactivation of IBa was produced by 1s prepulse and recovery determined by a brief test pulse after the indicated interval. This was repeated once every 60s (see inset above graph for voltage-protocol). Current amplitude during the recovery test pulse was normalized to peak IBa evoked by the prepulse. The solid lines show double exponential fits to the data. (F) PMA prolonged recovery from “slow inactivation”. Inactivation of IBa was produced by 10s prepulse and recovery was tracked using a series of brief test pulses applied at the indicated time points following the 10s prepulse (see inset). This was repeated twice in the same cell, once before application of PMA ((Ctl) and once in the presence of PMA (after 5-min exposure). Solid lines show double exponential fits to the mean data (control A1 = 0.23, A2 = 0.73, t1 = 1.21 s, t2 = 18.96 s; PMA A1 = 0.12, A2 = 0.76, t1 = 1.06 s, t2 = 49.2 s, fit comparison F = 27.6 p < 0.0001). The inset bar graph shows the mean time-constant for the slow phase of recovery calculated from fits to the individual cells (*** p = 0.001, paired t-test).
Mentions: We set out to investigate voltage-dependent inactivation of CaV2.2 Ca2+ channels and how it might be regulated by the PKC-activator PMA. To isolate voltage-dependent inactivation, we used barium rather than calcium as the extracellular divalent cation and included BAPTA (10 mM) in the intracellular patch-pipette solution to prevent calcium-dependent inactivation. G1A1 cells stably express CaV2.2, α2δ and β1b calcium channel subunits. Under our recording conditions, acute application of PMA (200 nM for 5-minutes) had no effect on the peak barium current (IBa) amplitude (Fig 1), and did not shift the current-voltage-relationship (Fig 1B), the voltage-dependence of inactivation (Fig 1C), or the rate/extent of inactivation during a 1s step depolarization (Fig 1D). To assess recovery of IBa from “fast” inactivation, we used a standard protocol in which a 1s prepulse (used to produce inactivation) was followed by a short test pulse to determine the extent of recovery (Fig 1E). This double pulse protocol was repeated every 60s and the recovery interval following the prepulse increased with each stimulus. Recovery following the 1s step was relatively fast (~ 50% recovery within 1s). Due to concerns with rundown and the possibility of a much slower component to the recovery, we limited this protocol to five repeats, covering the first 6-seconds of recovery. These data were fit well with a double exponential (Fig 1E) and neither of the fitted time constants were significantly changed in PMA treated cells (tau fast = 167 ± 12 ms in controls Vs. 161 ± 6 ms in PMA treated cells, p = 0.67 unpaired t-test: tau slow = 2.46 ± 0.49 s in controls Vs 3.1 ± 0.21 s in PMA treated cells, p = 0.23 unpaired t test).

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