Mutation of protein kinase C phosphorylation site S1076 on α-subunits affects BKCa channel activity in HEK-293 cells

Shu Zhu, Darren D Browning, Richard E. White, David J Fulton, Scott A Barman

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9 Citations (Scopus)

Abstract

Large conductance, calcium- and voltage-activated potassium (BK Ca) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BKCa channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BKCa channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BKCa channel α-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BKCa channels. Results showed that mutating S1076 altered the effect of PKC activation on BKCa channels in HEK-293 cells. Specifically, the phospho-deficient mutation BKCa-α(S1076A)/ β1 attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BKCa channels, whereas the phospho-mimetic mutation BKCa-α(S1076E)/β1 increased the excitatory effect of PMA on BKCa channels. In addition, the phosphonull mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BKCa channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BK Ca channel α-subunits influences BKCa channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume297
Issue number4
DOIs
StatePublished - Oct 14 2009

Fingerprint

HEK293 Cells
Serine
Protein Kinase C
Phosphorylation
Large-Conductance Calcium-Activated Potassium Channels
Cyclic GMP-Dependent Protein Kinases
Mutation
Tetradecanoylphorbol Acetate
Vascular Smooth Muscle
Lung
Membrane Potentials
Protein Kinases
Smooth Muscle
Potassium
Calcium
Kidney

Keywords

  • Channel subunits

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

Cite this

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title = "Mutation of protein kinase C phosphorylation site S1076 on α-subunits affects BKCa channel activity in HEK-293 cells",
abstract = "Large conductance, calcium- and voltage-activated potassium (BK Ca) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BKCa channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BKCa channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BKCa channel α-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BKCa channels. Results showed that mutating S1076 altered the effect of PKC activation on BKCa channels in HEK-293 cells. Specifically, the phospho-deficient mutation BKCa-α(S1076A)/ β1 attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BKCa channels, whereas the phospho-mimetic mutation BKCa-α(S1076E)/β1 increased the excitatory effect of PMA on BKCa channels. In addition, the phosphonull mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BKCa channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BK Ca channel α-subunits influences BKCa channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.",
keywords = "Channel subunits",
author = "Shu Zhu and Browning, {Darren D} and White, {Richard E.} and Fulton, {David J} and Barman, {Scott A}",
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T1 - Mutation of protein kinase C phosphorylation site S1076 on α-subunits affects BKCa channel activity in HEK-293 cells

AU - Zhu, Shu

AU - Browning, Darren D

AU - White, Richard E.

AU - Fulton, David J

AU - Barman, Scott A

PY - 2009/10/14

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N2 - Large conductance, calcium- and voltage-activated potassium (BK Ca) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BKCa channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BKCa channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BKCa channel α-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BKCa channels. Results showed that mutating S1076 altered the effect of PKC activation on BKCa channels in HEK-293 cells. Specifically, the phospho-deficient mutation BKCa-α(S1076A)/ β1 attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BKCa channels, whereas the phospho-mimetic mutation BKCa-α(S1076E)/β1 increased the excitatory effect of PMA on BKCa channels. In addition, the phosphonull mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BKCa channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BK Ca channel α-subunits influences BKCa channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.

AB - Large conductance, calcium- and voltage-activated potassium (BK Ca) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BKCa channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BKCa channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BKCa channel α-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BKCa channels. Results showed that mutating S1076 altered the effect of PKC activation on BKCa channels in HEK-293 cells. Specifically, the phospho-deficient mutation BKCa-α(S1076A)/ β1 attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BKCa channels, whereas the phospho-mimetic mutation BKCa-α(S1076E)/β1 increased the excitatory effect of PMA on BKCa channels. In addition, the phosphonull mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BKCa channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BK Ca channel α-subunits influences BKCa channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.

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