Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH(3) cells |
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Authors: | Wu Sheng-Nan Chang Han-Dong |
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Affiliation: | Department of Physiology, National Cheng Kung University Medical College, No 1, University Road, Tainan 70101, Taiwan. snwu@mail.ncku.edu.tw |
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Abstract: | The ATP-sensitive K(+) (K(ATP)) 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 K(ATP)-channel activity in pituitary GH(3) 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 K(ATP)-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 K(ATP)-channel activity in a concentration-dependent manner with an EC(50) value of 16 microM. DEPC-stimulated K(ATP)-channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH(3) 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 GH(3) cells. Simulation studies also demonstrated that the increased conductance of K(ATP)-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca(2+). The results indicate that chemical modification with DEPC enhances K(ATP)-channel activity and influences functional activities of pituitary GH(3) cells. |
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Keywords: | DEPC, diethyl pyrocarbonate KATP-channel, ATP-sensitive K+ channel [Ca2+]i, intracellular Ca2+ [Ca2+]ER, Ca2+ in the endoplasmic reticulum erg, ether-à-go-go-related gene I-V, current-voltage |
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