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

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 Ca2+ measurements revealed the ability of DEPC to decrease Ca2+ 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.