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Steady-state modulation of voltage-gated K+ channels in rat arterial smooth muscle by cyclic AMP-dependent protein kinase and protein phosphatase 2B.

Brignell JL, Perry MD, Nelson CP, Willets JM, Challiss RA, Davies NW - PLoS ONE (2015)

Bottom Line: Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents.Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents.These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom.

ABSTRACT
Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone.

No MeSH data available.


Related in: MedlinePlus

PKA activation of Kv currents remained following disruption of PKA-AKAP interactions.(A) Representative Kv current traces obtained immediately after establishing whole-cell recording (< 1 min Ht-31) and 10 min following establishment of the whole-cell configuration with 20 μM Ht-31 in the patch pipette. (B) Mean (± s.e.m.) I-V plots (current density normalized to cell capacitance) immediately after establishing whole-cell recording and 10 min after establishing whole cell configuration in the presence of Ht-31 (20 μM) in the patch pipette (n = 5). (C) Representative KATP current traces (normalized to cell capacitance) following the application of 100 nM isoprenaline in the absence or presence of 20 μM Ht-31 in the patch pipette; the current increase in response to isoprenaline is indicated by the dashed lines and arrow. (D) Mean glibeclamide-sensitive current (normalized to cell capacitance) following application of 100 nM isoprenaline in the absence or presence of 20 μM Ht-31 (n = 8 and 5 cells, respectively; *P<0.05; one-way ANOVA, Bonferroni’s post hoc test).
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pone.0121285.g006: PKA activation of Kv currents remained following disruption of PKA-AKAP interactions.(A) Representative Kv current traces obtained immediately after establishing whole-cell recording (< 1 min Ht-31) and 10 min following establishment of the whole-cell configuration with 20 μM Ht-31 in the patch pipette. (B) Mean (± s.e.m.) I-V plots (current density normalized to cell capacitance) immediately after establishing whole-cell recording and 10 min after establishing whole cell configuration in the presence of Ht-31 (20 μM) in the patch pipette (n = 5). (C) Representative KATP current traces (normalized to cell capacitance) following the application of 100 nM isoprenaline in the absence or presence of 20 μM Ht-31 in the patch pipette; the current increase in response to isoprenaline is indicated by the dashed lines and arrow. (D) Mean glibeclamide-sensitive current (normalized to cell capacitance) following application of 100 nM isoprenaline in the absence or presence of 20 μM Ht-31 (n = 8 and 5 cells, respectively; *P<0.05; one-way ANOVA, Bonferroni’s post hoc test).

Mentions: Localized PKA signalling is mediated by AKAP binding to the regulatory subunit of PKA [35], and this interaction can be abolished by Ht-31, a peptide that inhibits all PKA-AKAP interactions [36]. PKA dependent activation of KATP channels in MASMC by calcitonin gene-related peptide or by db-cAMP has been shown to be disrupted by inclusion of Ht-31 in the patch pipette [28]. We have shown already that there is considerable tonic PKA signalling maintaining the activity of Kv channels in these cells. If this is reliant on a PKA-AKAP interaction, inclusion of Ht-31 in the patch pipette should disrupt this, leading to a decline in Kv current following the establishment of the whole-cell configuration. However, we found no decline in the Kv current up to 10 min after the establishment of whole-cell configurations with Ht-31 (20 μM) in the pipette (Fig. 6; n = 5). In contrast, Ht-31 (20 μM) prevented the enhancement of the KATP current following application of isoprenaline (100 nM, see Fig. 6C and D). These data indicate that the interaction between PKA and AKAP, which is necessary to enable PKA signalling to KATP channels, may not be involved in PKA-mediated enhancement of Kv currents in MASMC.


Steady-state modulation of voltage-gated K+ channels in rat arterial smooth muscle by cyclic AMP-dependent protein kinase and protein phosphatase 2B.

Brignell JL, Perry MD, Nelson CP, Willets JM, Challiss RA, Davies NW - PLoS ONE (2015)

PKA activation of Kv currents remained following disruption of PKA-AKAP interactions.(A) Representative Kv current traces obtained immediately after establishing whole-cell recording (< 1 min Ht-31) and 10 min following establishment of the whole-cell configuration with 20 μM Ht-31 in the patch pipette. (B) Mean (± s.e.m.) I-V plots (current density normalized to cell capacitance) immediately after establishing whole-cell recording and 10 min after establishing whole cell configuration in the presence of Ht-31 (20 μM) in the patch pipette (n = 5). (C) Representative KATP current traces (normalized to cell capacitance) following the application of 100 nM isoprenaline in the absence or presence of 20 μM Ht-31 in the patch pipette; the current increase in response to isoprenaline is indicated by the dashed lines and arrow. (D) Mean glibeclamide-sensitive current (normalized to cell capacitance) following application of 100 nM isoprenaline in the absence or presence of 20 μM Ht-31 (n = 8 and 5 cells, respectively; *P<0.05; one-way ANOVA, Bonferroni’s post hoc test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4368632&req=5

pone.0121285.g006: PKA activation of Kv currents remained following disruption of PKA-AKAP interactions.(A) Representative Kv current traces obtained immediately after establishing whole-cell recording (< 1 min Ht-31) and 10 min following establishment of the whole-cell configuration with 20 μM Ht-31 in the patch pipette. (B) Mean (± s.e.m.) I-V plots (current density normalized to cell capacitance) immediately after establishing whole-cell recording and 10 min after establishing whole cell configuration in the presence of Ht-31 (20 μM) in the patch pipette (n = 5). (C) Representative KATP current traces (normalized to cell capacitance) following the application of 100 nM isoprenaline in the absence or presence of 20 μM Ht-31 in the patch pipette; the current increase in response to isoprenaline is indicated by the dashed lines and arrow. (D) Mean glibeclamide-sensitive current (normalized to cell capacitance) following application of 100 nM isoprenaline in the absence or presence of 20 μM Ht-31 (n = 8 and 5 cells, respectively; *P<0.05; one-way ANOVA, Bonferroni’s post hoc test).
Mentions: Localized PKA signalling is mediated by AKAP binding to the regulatory subunit of PKA [35], and this interaction can be abolished by Ht-31, a peptide that inhibits all PKA-AKAP interactions [36]. PKA dependent activation of KATP channels in MASMC by calcitonin gene-related peptide or by db-cAMP has been shown to be disrupted by inclusion of Ht-31 in the patch pipette [28]. We have shown already that there is considerable tonic PKA signalling maintaining the activity of Kv channels in these cells. If this is reliant on a PKA-AKAP interaction, inclusion of Ht-31 in the patch pipette should disrupt this, leading to a decline in Kv current following the establishment of the whole-cell configuration. However, we found no decline in the Kv current up to 10 min after the establishment of whole-cell configurations with Ht-31 (20 μM) in the pipette (Fig. 6; n = 5). In contrast, Ht-31 (20 μM) prevented the enhancement of the KATP current following application of isoprenaline (100 nM, see Fig. 6C and D). These data indicate that the interaction between PKA and AKAP, which is necessary to enable PKA signalling to KATP channels, may not be involved in PKA-mediated enhancement of Kv currents in MASMC.

Bottom Line: Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents.Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents.These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom.

ABSTRACT
Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone.

No MeSH data available.


Related in: MedlinePlus