Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH3 cells

Sheng-Nan Wu, Han Dong Chang

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The ATP-sensitive K+ (KATP) channels are composed of sulfonylurea receptor and inwardly rectifying K+ channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies KATP-channel activity in pituitary GH3 cells. Steady-state fluctuation analyses of macroscopic K+ current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of KATP-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance KATP-channel activity in a concentration-dependent manner with an EC50 value of 16 μM. DEPC-stimulated KATP- channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH3 cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca 2+ measurements revealed the ability of DEPC to decrease Ca 2+ oscillations in GH3 cells. Simulation studies also demonstrated that the increased conductance of KATP-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca2+. The results indicate that chemical modification with DEPC enhances KATP-channel activity and influences functional activities of pituitary GH3 cells.

Original languageEnglish
Pages (from-to)615-623
Number of pages9
JournalBiochemical Pharmacology
Volume71
Issue number5
DOIs
Publication statusPublished - 2006 Feb 28

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Diethyl Pyrocarbonate
KATP Channels
Histidine
Action Potentials
Glyburide
Adenosine Triphosphate
Sulfonylurea Receptors
Inwardly Rectifying Potassium Channel
Clamping devices
Chemical modification
Power spectrum
Metabolism
Adenosine Diphosphate

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Pharmacology

Cite this

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title = "Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH3 cells",
abstract = "The ATP-sensitive K+ (KATP) channels are composed of sulfonylurea receptor and inwardly rectifying K+ channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies KATP-channel activity in pituitary GH3 cells. Steady-state fluctuation analyses of macroscopic K+ current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of KATP-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance KATP-channel activity in a concentration-dependent manner with an EC50 value of 16 μM. DEPC-stimulated KATP- channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH3 cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca 2+ measurements revealed the ability of DEPC to decrease Ca 2+ oscillations in GH3 cells. Simulation studies also demonstrated that the increased conductance of KATP-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca2+. The results indicate that chemical modification with DEPC enhances KATP-channel activity and influences functional activities of pituitary GH3 cells.",
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Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH3 cells. / Wu, Sheng-Nan; Chang, Han Dong.

In: Biochemical Pharmacology, Vol. 71, No. 5, 28.02.2006, p. 615-623.

Research output: Contribution to journalArticle

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AU - Wu, Sheng-Nan

AU - Chang, Han Dong

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N2 - The ATP-sensitive K+ (KATP) channels are composed of sulfonylurea receptor and inwardly rectifying K+ channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies KATP-channel activity in pituitary GH3 cells. Steady-state fluctuation analyses of macroscopic K+ current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of KATP-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance KATP-channel activity in a concentration-dependent manner with an EC50 value of 16 μM. DEPC-stimulated KATP- channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH3 cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca 2+ measurements revealed the ability of DEPC to decrease Ca 2+ oscillations in GH3 cells. Simulation studies also demonstrated that the increased conductance of KATP-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca2+. The results indicate that chemical modification with DEPC enhances KATP-channel activity and influences functional activities of pituitary GH3 cells.

AB - The ATP-sensitive K+ (KATP) channels are composed of sulfonylurea receptor and inwardly rectifying K+ channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies KATP-channel activity in pituitary GH3 cells. Steady-state fluctuation analyses of macroscopic K+ current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of KATP-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance KATP-channel activity in a concentration-dependent manner with an EC50 value of 16 μM. DEPC-stimulated KATP- channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH3 cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca 2+ measurements revealed the ability of DEPC to decrease Ca 2+ oscillations in GH3 cells. Simulation studies also demonstrated that the increased conductance of KATP-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca2+. The results indicate that chemical modification with DEPC enhances KATP-channel activity and influences functional activities of pituitary GH3 cells.

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