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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 prolonged recovery of ICa from inactivation in adrenal chromaffin cells.(A–C) Chromaffin cells were stimulated with a 10s step depolarization and recovery from inactivation tracked at the indicated time points. ICa amplitude (normalized to that before the 10 step depolarization) is plotted for each of the recovery time points and the mean data fit with a double exponential (solid lines). A) Control cells with no PMA treatment (n = 11) (fit parameters: control Y0 = 0.07, A1 = 0.29, A2 = 0.49, t1 = 1.5 s, t2 = 32.1 s; control repeat Y0 = 0.07, A1 = 0.36, A2 = 0.37, t1 = 1.5 s, t2 = 52.6 s: fit comparison F = 1.66 p = 0.21). B) Control cells with PMA (n = 6) (fit parameters: control Y0 = 0.05, A1 = 0.28, A2 = 0.56, t1 = 0.7 s, t2 = 23.5 s; PMA Y0 = 0.03, A1 = 0.24, A2 = 0.55, t1 = 1.1 s, t2 = 69.6 s, comparison of fits F = 35.2 p < 0.0001). C)Cells pretreated with calphostin C (n = 6) (fit parameters: calphostin Y0 = 0.08, A1 = 0.31, A2 = 0.52, t1 = 1.2 s, t2 = 22.6 s; calphostin + PMA Y0 = 0.10, A1 = 0.36, A2 = 0.45, t1 = 1.0 s, t2 = 52.7 s, comparison of fits F = 1.73 p = 0.19). (D) The percent inhibition of recovery at the 40 s time point was calculated (R40—see results section for more detail). The bar graph shows R40 produced by PMA in control cells (PMA; n = 6), cells pretreated with bisindolylmaleimide-1 (Bis; n = 4), and cells pretreated with calphostin C (Cal; n = 6). The R40 for control cells stimulated twice in the absence of PMA is also shown (ctl/ctl; n = 11). 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).
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pone.0134117.g007: PMA prolonged recovery of ICa from inactivation in adrenal chromaffin cells.(A–C) Chromaffin cells were stimulated with a 10s step depolarization and recovery from inactivation tracked at the indicated time points. ICa amplitude (normalized to that before the 10 step depolarization) is plotted for each of the recovery time points and the mean data fit with a double exponential (solid lines). A) Control cells with no PMA treatment (n = 11) (fit parameters: control Y0 = 0.07, A1 = 0.29, A2 = 0.49, t1 = 1.5 s, t2 = 32.1 s; control repeat Y0 = 0.07, A1 = 0.36, A2 = 0.37, t1 = 1.5 s, t2 = 52.6 s: fit comparison F = 1.66 p = 0.21). B) Control cells with PMA (n = 6) (fit parameters: control Y0 = 0.05, A1 = 0.28, A2 = 0.56, t1 = 0.7 s, t2 = 23.5 s; PMA Y0 = 0.03, A1 = 0.24, A2 = 0.55, t1 = 1.1 s, t2 = 69.6 s, comparison of fits F = 35.2 p < 0.0001). C)Cells pretreated with calphostin C (n = 6) (fit parameters: calphostin Y0 = 0.08, A1 = 0.31, A2 = 0.52, t1 = 1.2 s, t2 = 22.6 s; calphostin + PMA Y0 = 0.10, A1 = 0.36, A2 = 0.45, t1 = 1.0 s, t2 = 52.7 s, comparison of fits F = 1.73 p = 0.19). (D) The percent inhibition of recovery at the 40 s time point was calculated (R40—see results section for more detail). The bar graph shows R40 produced by PMA in control cells (PMA; n = 6), cells pretreated with bisindolylmaleimide-1 (Bis; n = 4), and cells pretreated with calphostin C (Cal; n = 6). The R40 for control cells stimulated twice in the absence of PMA is also shown (ctl/ctl; n = 11). 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).

Mentions: “Fast” voltage-dependent inactivation is minimal in chromaffin cells, possibly due to expression of the β2a subunit [58, 59]. However, the 10s step depolarization paradigm produced robust inactivation of ICa both in control conditions (95 ± 1%) and in the presence of PMA (97 ± 0.5%, n = 7; p = 0.132, paired t-test). Recovery from inactivation exhibited at least two kinetic components (Fig 7). PMA clearly prolonged recovery from inactivation, with a particularly prominent effect on the slower time constant which was increased from 26.6 ± 1.4 s to 90.9 ± 8.8 s (n = 7, p = 0.0002, paired t-test). The R40 (% change in recovery at 40s) showed that PMA inhibited recovery by 35.5 ± 1.6% (n = 7). Pretreating cells with bisindolylmaleimide-1 modestly reduced R40 to 21.6 ± 7% (n = 5) although this was not statistically significant, and calphostin C significantly reduced R40 to 12.8 ± 4% (n = 6; p < 0.01 one-way ANOVA followed by Dunnetts post-test) (Fig 7D). These data mirror those obtained in recombinant channels and confirmed that PMA prolongs recovery from “slow” inactivation for endogenously expressed ICa in neuroendocrine cells.


"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 prolonged recovery of ICa from inactivation in adrenal chromaffin cells.(A–C) Chromaffin cells were stimulated with a 10s step depolarization and recovery from inactivation tracked at the indicated time points. ICa amplitude (normalized to that before the 10 step depolarization) is plotted for each of the recovery time points and the mean data fit with a double exponential (solid lines). A) Control cells with no PMA treatment (n = 11) (fit parameters: control Y0 = 0.07, A1 = 0.29, A2 = 0.49, t1 = 1.5 s, t2 = 32.1 s; control repeat Y0 = 0.07, A1 = 0.36, A2 = 0.37, t1 = 1.5 s, t2 = 52.6 s: fit comparison F = 1.66 p = 0.21). B) Control cells with PMA (n = 6) (fit parameters: control Y0 = 0.05, A1 = 0.28, A2 = 0.56, t1 = 0.7 s, t2 = 23.5 s; PMA Y0 = 0.03, A1 = 0.24, A2 = 0.55, t1 = 1.1 s, t2 = 69.6 s, comparison of fits F = 35.2 p < 0.0001). C)Cells pretreated with calphostin C (n = 6) (fit parameters: calphostin Y0 = 0.08, A1 = 0.31, A2 = 0.52, t1 = 1.2 s, t2 = 22.6 s; calphostin + PMA Y0 = 0.10, A1 = 0.36, A2 = 0.45, t1 = 1.0 s, t2 = 52.7 s, comparison of fits F = 1.73 p = 0.19). (D) The percent inhibition of recovery at the 40 s time point was calculated (R40—see results section for more detail). The bar graph shows R40 produced by PMA in control cells (PMA; n = 6), cells pretreated with bisindolylmaleimide-1 (Bis; n = 4), and cells pretreated with calphostin C (Cal; n = 6). The R40 for control cells stimulated twice in the absence of PMA is also shown (ctl/ctl; n = 11). 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).
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Related In: Results  -  Collection

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pone.0134117.g007: PMA prolonged recovery of ICa from inactivation in adrenal chromaffin cells.(A–C) Chromaffin cells were stimulated with a 10s step depolarization and recovery from inactivation tracked at the indicated time points. ICa amplitude (normalized to that before the 10 step depolarization) is plotted for each of the recovery time points and the mean data fit with a double exponential (solid lines). A) Control cells with no PMA treatment (n = 11) (fit parameters: control Y0 = 0.07, A1 = 0.29, A2 = 0.49, t1 = 1.5 s, t2 = 32.1 s; control repeat Y0 = 0.07, A1 = 0.36, A2 = 0.37, t1 = 1.5 s, t2 = 52.6 s: fit comparison F = 1.66 p = 0.21). B) Control cells with PMA (n = 6) (fit parameters: control Y0 = 0.05, A1 = 0.28, A2 = 0.56, t1 = 0.7 s, t2 = 23.5 s; PMA Y0 = 0.03, A1 = 0.24, A2 = 0.55, t1 = 1.1 s, t2 = 69.6 s, comparison of fits F = 35.2 p < 0.0001). C)Cells pretreated with calphostin C (n = 6) (fit parameters: calphostin Y0 = 0.08, A1 = 0.31, A2 = 0.52, t1 = 1.2 s, t2 = 22.6 s; calphostin + PMA Y0 = 0.10, A1 = 0.36, A2 = 0.45, t1 = 1.0 s, t2 = 52.7 s, comparison of fits F = 1.73 p = 0.19). (D) The percent inhibition of recovery at the 40 s time point was calculated (R40—see results section for more detail). The bar graph shows R40 produced by PMA in control cells (PMA; n = 6), cells pretreated with bisindolylmaleimide-1 (Bis; n = 4), and cells pretreated with calphostin C (Cal; n = 6). The R40 for control cells stimulated twice in the absence of PMA is also shown (ctl/ctl; n = 11). 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).
Mentions: “Fast” voltage-dependent inactivation is minimal in chromaffin cells, possibly due to expression of the β2a subunit [58, 59]. However, the 10s step depolarization paradigm produced robust inactivation of ICa both in control conditions (95 ± 1%) and in the presence of PMA (97 ± 0.5%, n = 7; p = 0.132, paired t-test). Recovery from inactivation exhibited at least two kinetic components (Fig 7). PMA clearly prolonged recovery from inactivation, with a particularly prominent effect on the slower time constant which was increased from 26.6 ± 1.4 s to 90.9 ± 8.8 s (n = 7, p = 0.0002, paired t-test). The R40 (% change in recovery at 40s) showed that PMA inhibited recovery by 35.5 ± 1.6% (n = 7). Pretreating cells with bisindolylmaleimide-1 modestly reduced R40 to 21.6 ± 7% (n = 5) although this was not statistically significant, and calphostin C significantly reduced R40 to 12.8 ± 4% (n = 6; p < 0.01 one-way ANOVA followed by Dunnetts post-test) (Fig 7D). These data mirror those obtained in recombinant channels and confirmed that PMA prolongs recovery from “slow” inactivation for endogenously expressed ICa in neuroendocrine cells.

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