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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

Testing the involvement of PKC in the prolonged recovery from inactivation produced by PMA.(A) Cells were pretreated with calphostin C (200 nM) or a mixture of bisindolylmaleimide-1 (Bis; 500 nM) + Go6983 (100 nM). Inactivation was produced by a stimulus train (5Hz for 10s as in Fig 4) and the fractional recovery from inactivation determined first in the absence and then in the presence of PMA. Solid lines show an exponential fit to the mean data (left panel: calphostin A = 0.95, t = 17.2 s; calphostin + PMA A = 1.0, t = 23.7 s, comparison of fits F = 3.62 p = 0.06: right panel: Bis/Go A = 0.80, t = 15.6 s; Bis/Go + PMA A = 0.67 t = 40.7 s, comparison of fits F = 25.3 p < 0.0001). The arrows labeled “R40“denote the 40s recovery time point. (B) Plots the mean recovery time constant determined from an exponential fit in each cell before (left panel) and during (right panel) application of PMA, or the inactive control 4α-PMA. “Control” = no pretreatment (n = 6); “PKCi” = cells recorded with intracellular application of a pseudosubstrate peptide inhibitor of PKC (2 μM PKC(19–36) (n = 6); “Bis + Go” = cells pretreated with bisindolylmaleimide-1 (500nM) + Go6983 (100nM) (n = 6); “Calphos” = cells pretreated with calphostin C (200 nM) (n = 7). Also shown is data for cells treated with 4α-PMA (n = 4). Pretreatment with the various drugs did not significantly alter the recovery time constant before application of PMA (left panel). The effect of PMA was significantly reduced by pretreatment with Bis + Go or calphostin C (right panel) (ns, not significantly different, * p < 0.05, *** P < 0.001 compared to the control PMA cells (red bar) determined using one-way ANOVA and Dunnett’s post-test). (C) To quantify the change in recovery rate produced by PMA, a ratio of the recovery time constants was calculated in each cell (tau in the presence of PMA / tau before application of PMA). Statistical significance compared to the control PMA cells (red bar) was determined using one-way ANOVA and Dunnett’s post-test (ns, not significantly different, ** P < 0.01). (D) Another index to compare the various drug treatments is the percent inhibition of recovery at the 40 s time point (R40—see panel A and results section for more detail). Statistical significance compared to the control PMA cells (red bar) was determined using one-way ANOVA and Dunnett’s post-test (ns, not significantly different, * p < 0.05, *** P < 0.001).
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pone.0134117.g005: Testing the involvement of PKC in the prolonged recovery from inactivation produced by PMA.(A) Cells were pretreated with calphostin C (200 nM) or a mixture of bisindolylmaleimide-1 (Bis; 500 nM) + Go6983 (100 nM). Inactivation was produced by a stimulus train (5Hz for 10s as in Fig 4) and the fractional recovery from inactivation determined first in the absence and then in the presence of PMA. Solid lines show an exponential fit to the mean data (left panel: calphostin A = 0.95, t = 17.2 s; calphostin + PMA A = 1.0, t = 23.7 s, comparison of fits F = 3.62 p = 0.06: right panel: Bis/Go A = 0.80, t = 15.6 s; Bis/Go + PMA A = 0.67 t = 40.7 s, comparison of fits F = 25.3 p < 0.0001). The arrows labeled “R40“denote the 40s recovery time point. (B) Plots the mean recovery time constant determined from an exponential fit in each cell before (left panel) and during (right panel) application of PMA, or the inactive control 4α-PMA. “Control” = no pretreatment (n = 6); “PKCi” = cells recorded with intracellular application of a pseudosubstrate peptide inhibitor of PKC (2 μM PKC(19–36) (n = 6); “Bis + Go” = cells pretreated with bisindolylmaleimide-1 (500nM) + Go6983 (100nM) (n = 6); “Calphos” = cells pretreated with calphostin C (200 nM) (n = 7). Also shown is data for cells treated with 4α-PMA (n = 4). Pretreatment with the various drugs did not significantly alter the recovery time constant before application of PMA (left panel). The effect of PMA was significantly reduced by pretreatment with Bis + Go or calphostin C (right panel) (ns, not significantly different, * p < 0.05, *** P < 0.001 compared to the control PMA cells (red bar) determined using one-way ANOVA and Dunnett’s post-test). (C) To quantify the change in recovery rate produced by PMA, a ratio of the recovery time constants was calculated in each cell (tau in the presence of PMA / tau before application of PMA). Statistical significance compared to the control PMA cells (red bar) was determined using one-way ANOVA and Dunnett’s post-test (ns, not significantly different, ** P < 0.01). (D) Another index to compare the various drug treatments is the percent inhibition of recovery at the 40 s time point (R40—see panel A and results section for more detail). Statistical significance compared to the control PMA cells (red bar) was determined using one-way ANOVA and Dunnett’s post-test (ns, not significantly different, * p < 0.05, *** P < 0.001).

Mentions: 4α-PMA is an inactive control for PMA that does not activate PKC. We found that 4α-PMA did not significantly alter recovery from inactivation following a 5Hz/10s stimulus train in HEK cells expressing CaV2.2, β1b and α2δ, (tau control = 19.8 ± 3.1 s Vs tau 4α-phorbol = 22.5 ± 4.7 s; n = 4; p = 0.28, paired t-test). This is consistent with the involvement of PKC, so we tested several PKC inhibitors to see if they blocked the effect of PMA on recovery from inactivation. Cells were pre-incubated for 20–30 minutes with the inhibitors and then stimulated first in the presence of the inhibitor alone and then in the presence of the inhibitor + PMA. The inhibitors used were PKC(19–36), a pseudosubstrate peptide inhibitor of PKC that was added to the intracellular patch-pipette solution (2 μM); a combination of bisindolylmaleimide-1 (500nM) + Go6983 (100nM); calphostin C (200 nM). Recovery from inactivation following a 5Hz/10s train was fit with an exponential to determine the recovery time constant (Fig 5A and 5B). None of the drug pretreatments significantly altered the baseline recovery rate prior to application of PMA (Fig 5B) (p = 0.09, F = 2.28; one-way ANOVA followed by Dunnett’s post-test). However, the recovery time constant in the presence of PMA was significantly reduced by pretreatment with bisindolylmaleimide-1 + Go6983 or calphostin C (p < 0.0001, F = 11.64; one-way ANOVA followed by Dunnett’s post-test for multiple pairwise comparisons) (Fig 5B). To compare the various treatment groups, the change in recovery time constant was expressed as a ratio (tau in the presence of PMA / tau before PMA) (Fig 5C). The slowing of recovery produced by PMA (tau ratio) was not seen with the control 4α-PMA, was abolished by pretreatment with calphostin C, and partially, but not significantly reduced in cells pretreated with PKC(19–36), or bisindolylmaleimide-I + Go6983 (p = 0.005, F = 4.885, one-way ANOVA, followed by Dunnett’s post-test). It has been reported previously that the extent, but not the rate, of CaV2.2 recovery from inactivation can be modulated by alternative splicing [37]. Therefore, we calculated the percent change in recovery produced by PMA at the 40 s time point (R40) (Fig 4A and 4C). In control cells (before application of PMA) the fractional recovery at this time was 0.67 ± 0.07, and in the presence of PMA this was reduced to 0.23 ±0.02 (n = 6, p = 0.0026, paired t-test) resulting in a calculated R40 of 61 ± 6.9%. We calculated R40 for the various treatment groups (different PKC inhibitors) and found it was significantly reduced by bisindolylmaleimeide-1 + Go6983 and abolished by calphostin C (Fig 5D) (p = 0.0001, F = 29.3, one-way ANOVA followed by Dunnett’s post-test). Thus, our data show that the effect of PMA on recovery from inactivation was abolished by calphostin C and partially blocked by bisindolylmaleimeide-1 + Go6983.


"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)

Testing the involvement of PKC in the prolonged recovery from inactivation produced by PMA.(A) Cells were pretreated with calphostin C (200 nM) or a mixture of bisindolylmaleimide-1 (Bis; 500 nM) + Go6983 (100 nM). Inactivation was produced by a stimulus train (5Hz for 10s as in Fig 4) and the fractional recovery from inactivation determined first in the absence and then in the presence of PMA. Solid lines show an exponential fit to the mean data (left panel: calphostin A = 0.95, t = 17.2 s; calphostin + PMA A = 1.0, t = 23.7 s, comparison of fits F = 3.62 p = 0.06: right panel: Bis/Go A = 0.80, t = 15.6 s; Bis/Go + PMA A = 0.67 t = 40.7 s, comparison of fits F = 25.3 p < 0.0001). The arrows labeled “R40“denote the 40s recovery time point. (B) Plots the mean recovery time constant determined from an exponential fit in each cell before (left panel) and during (right panel) application of PMA, or the inactive control 4α-PMA. “Control” = no pretreatment (n = 6); “PKCi” = cells recorded with intracellular application of a pseudosubstrate peptide inhibitor of PKC (2 μM PKC(19–36) (n = 6); “Bis + Go” = cells pretreated with bisindolylmaleimide-1 (500nM) + Go6983 (100nM) (n = 6); “Calphos” = cells pretreated with calphostin C (200 nM) (n = 7). Also shown is data for cells treated with 4α-PMA (n = 4). Pretreatment with the various drugs did not significantly alter the recovery time constant before application of PMA (left panel). The effect of PMA was significantly reduced by pretreatment with Bis + Go or calphostin C (right panel) (ns, not significantly different, * p < 0.05, *** P < 0.001 compared to the control PMA cells (red bar) determined using one-way ANOVA and Dunnett’s post-test). (C) To quantify the change in recovery rate produced by PMA, a ratio of the recovery time constants was calculated in each cell (tau in the presence of PMA / tau before application of PMA). Statistical significance compared to the control PMA cells (red bar) was determined using one-way ANOVA and Dunnett’s post-test (ns, not significantly different, ** P < 0.01). (D) Another index to compare the various drug treatments is the percent inhibition of recovery at the 40 s time point (R40—see panel A and results section for more detail). Statistical significance compared to the control PMA cells (red bar) was determined using one-way ANOVA and Dunnett’s post-test (ns, not significantly different, * p < 0.05, *** P < 0.001).
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pone.0134117.g005: Testing the involvement of PKC in the prolonged recovery from inactivation produced by PMA.(A) Cells were pretreated with calphostin C (200 nM) or a mixture of bisindolylmaleimide-1 (Bis; 500 nM) + Go6983 (100 nM). Inactivation was produced by a stimulus train (5Hz for 10s as in Fig 4) and the fractional recovery from inactivation determined first in the absence and then in the presence of PMA. Solid lines show an exponential fit to the mean data (left panel: calphostin A = 0.95, t = 17.2 s; calphostin + PMA A = 1.0, t = 23.7 s, comparison of fits F = 3.62 p = 0.06: right panel: Bis/Go A = 0.80, t = 15.6 s; Bis/Go + PMA A = 0.67 t = 40.7 s, comparison of fits F = 25.3 p < 0.0001). The arrows labeled “R40“denote the 40s recovery time point. (B) Plots the mean recovery time constant determined from an exponential fit in each cell before (left panel) and during (right panel) application of PMA, or the inactive control 4α-PMA. “Control” = no pretreatment (n = 6); “PKCi” = cells recorded with intracellular application of a pseudosubstrate peptide inhibitor of PKC (2 μM PKC(19–36) (n = 6); “Bis + Go” = cells pretreated with bisindolylmaleimide-1 (500nM) + Go6983 (100nM) (n = 6); “Calphos” = cells pretreated with calphostin C (200 nM) (n = 7). Also shown is data for cells treated with 4α-PMA (n = 4). Pretreatment with the various drugs did not significantly alter the recovery time constant before application of PMA (left panel). The effect of PMA was significantly reduced by pretreatment with Bis + Go or calphostin C (right panel) (ns, not significantly different, * p < 0.05, *** P < 0.001 compared to the control PMA cells (red bar) determined using one-way ANOVA and Dunnett’s post-test). (C) To quantify the change in recovery rate produced by PMA, a ratio of the recovery time constants was calculated in each cell (tau in the presence of PMA / tau before application of PMA). Statistical significance compared to the control PMA cells (red bar) was determined using one-way ANOVA and Dunnett’s post-test (ns, not significantly different, ** P < 0.01). (D) Another index to compare the various drug treatments is the percent inhibition of recovery at the 40 s time point (R40—see panel A and results section for more detail). Statistical significance compared to the control PMA cells (red bar) was determined using one-way ANOVA and Dunnett’s post-test (ns, not significantly different, * p < 0.05, *** P < 0.001).
Mentions: 4α-PMA is an inactive control for PMA that does not activate PKC. We found that 4α-PMA did not significantly alter recovery from inactivation following a 5Hz/10s stimulus train in HEK cells expressing CaV2.2, β1b and α2δ, (tau control = 19.8 ± 3.1 s Vs tau 4α-phorbol = 22.5 ± 4.7 s; n = 4; p = 0.28, paired t-test). This is consistent with the involvement of PKC, so we tested several PKC inhibitors to see if they blocked the effect of PMA on recovery from inactivation. Cells were pre-incubated for 20–30 minutes with the inhibitors and then stimulated first in the presence of the inhibitor alone and then in the presence of the inhibitor + PMA. The inhibitors used were PKC(19–36), a pseudosubstrate peptide inhibitor of PKC that was added to the intracellular patch-pipette solution (2 μM); a combination of bisindolylmaleimide-1 (500nM) + Go6983 (100nM); calphostin C (200 nM). Recovery from inactivation following a 5Hz/10s train was fit with an exponential to determine the recovery time constant (Fig 5A and 5B). None of the drug pretreatments significantly altered the baseline recovery rate prior to application of PMA (Fig 5B) (p = 0.09, F = 2.28; one-way ANOVA followed by Dunnett’s post-test). However, the recovery time constant in the presence of PMA was significantly reduced by pretreatment with bisindolylmaleimide-1 + Go6983 or calphostin C (p < 0.0001, F = 11.64; one-way ANOVA followed by Dunnett’s post-test for multiple pairwise comparisons) (Fig 5B). To compare the various treatment groups, the change in recovery time constant was expressed as a ratio (tau in the presence of PMA / tau before PMA) (Fig 5C). The slowing of recovery produced by PMA (tau ratio) was not seen with the control 4α-PMA, was abolished by pretreatment with calphostin C, and partially, but not significantly reduced in cells pretreated with PKC(19–36), or bisindolylmaleimide-I + Go6983 (p = 0.005, F = 4.885, one-way ANOVA, followed by Dunnett’s post-test). It has been reported previously that the extent, but not the rate, of CaV2.2 recovery from inactivation can be modulated by alternative splicing [37]. Therefore, we calculated the percent change in recovery produced by PMA at the 40 s time point (R40) (Fig 4A and 4C). In control cells (before application of PMA) the fractional recovery at this time was 0.67 ± 0.07, and in the presence of PMA this was reduced to 0.23 ±0.02 (n = 6, p = 0.0026, paired t-test) resulting in a calculated R40 of 61 ± 6.9%. We calculated R40 for the various treatment groups (different PKC inhibitors) and found it was significantly reduced by bisindolylmaleimeide-1 + Go6983 and abolished by calphostin C (Fig 5D) (p = 0.0001, F = 29.3, one-way ANOVA followed by Dunnett’s post-test). Thus, our data show that the effect of PMA on recovery from inactivation was abolished by calphostin C and partially blocked by bisindolylmaleimeide-1 + Go6983.

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