Slowing of ERG current deactivation in NG108-15 cells by the histidine-specific reagent diethylpyrocarbonate

Abstract

The aim of this study was to explore and characterize the effect of the histidine-specific reagent diethylpyrocarbonate (DEPC) on the ERG ( ether-à-go-go related gene) channels of whole-cell voltage-clamped NG108-15 neuroblastoma x glioma hybrid cells. The channels were fully activated by long depolarizing prepulses. Hyperpolarizing pulses elicited K + inward currents which deactivated after reaching a peak. DEPC (0.26–2.1 mM, externally applied for 5–12 min) irreversibly decreased τ −1, the rate constant of deactivation. At a pulse potential of −120 mV τ −1 decreased on average by 60%. The effect can be described as a −25 mV shift of the τ −1( V) curve. The activation curve and the curve relating steady-state current to pulse potential were shifted by similar amounts. The decrease of τ −1 was the same at 0.26 and at 2.1 mM, but developed faster at the higher concentration. The slowing of deactivation was only seen when the cells were held at a potential of −20 mV. At this potential it developed with a time constant of 47 s. At more negative holding potentials (−40 or −70 mV) only a slight reduction of the peak occurred. The observations suggest preferential binding of DEPC to the open and inactivated channel states. The DEPC effect can possibly be explained by the reaction of DEPC with histidine residues or other amino acids in the external loops of the channel. However, dichloro-(2,2′:6′,2″-terpyridine)-platinum (II) dihydrate (DTPD), another histidine-specific reagent, markedly decreased the peak current without affecting τ −1. Therefore, the possibility that (some of) the effects of DEPC and DTPD are unrelated to their property as histidine-specific reagents cannot be excluded.