The Inhibition of TREK2 Channel by an Oxidizing Agent, 5,5'-dithio-bis (2-nitrobenzoic acid), via Interaction with the C-terminus Distal to the 353rd Amino Acid

Abstract

TREK (TWIK-RElated K(+) channels) and TRAAK (TWIK-Related Arachidonic acid Activated K(+) channels) were expressed in COS-7 cells, and the channel activities were recorded from inside-out membrane patches using holding potential of -40 mV in symmetrical 150 mM K(+) solution. Intracellular application of an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), markedly decreased the activity of the TREK2, and the activity was partially reversed by the reducing agent, dithiothreitol (DTT). In order to examine the possibility that the target sites for the oxidizing agents might be located in the C-terminus of TREK2, two chimeras were constructed: TREK2 (1-383)/TASK3C and TREK2 (1-353)/TASK3C. The channel activity in the TREK2 (1-383)/TASK3C chimera was still inhibited by DTNB, but not in the TREK2 (1-353)/TASK3C chimera. These results indicate that TREK2 is inhibited by oxidation, and that the target site for oxidation is located between the amino acid residues 353 and 383 in the C-terminus of the TREK2 protein.

Keywords: 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB); C-terminus; K2P; Oxidizing agent; TREK2; Two-pore domain K+ channel; dithiothreitol (DTT).

Fig. 1

The effects of oxidizing chemical agent, DTNB, on TRAAK, TREK1, and TREK2 in inside-out patch configurations. (A) The left panel shows the general single channel activity without DTNB, and the right panel shows channel activity with DTNB (2 mM). DTNB was applied to the bath solution, and the holding potential was held at -40 mV. "Close" represents the closed-channel level, and "open" represents the open-channel level. (B) The relative channel activity with DTNB on each channel. (C) Dose response curve of DTNB on TREK2. The pipette and bath solution contained 150 mM KCl, 5 mM EGTA, 10 mM HEPES, and 1 mM MgCl₂. The asterisk indicates a significant difference from the respective control (p<0.05).

Fig. 2

The effect of extracellular application of DTNB (2 mM) and DTT (5 mM) on TREK2 using outside-out patch configurations. The holding potential was held at -40 mV. (A) The channel activity was not changed by DTNB and DTT. (B) Expanded scale trace of channel activity from A.

Fig. 3

The intracellular C-terminus of TREK2 modulates DTNB effects upon channel activity. (A) The first and second panels show the effect of DTNB (2 mM) on wild type TREK2 (1-538) and TREK2 chimera [TREK2 (1383)/TASK3C], respectively. The third panel shows the effect of DTNB on TREK2 chimera [TREK2 (1-353)/TASK3C]. The fourth panel shows the effect of DTNB on mutant TREK2 (1-383, M375L)/TASK3C (methionine-to-leucine substitution at position 375). (B) Expanded scale trace of channel activity from the A.

Fig. 4

The effect of intracellular acidic pH on wild type TREK2 and a chimera [TREK2 (1-353)/TASK3C] in the presence of DTNB. (A) An intracellular acidic pH activated wild type TREK2 in the absence of DTNB, but intracellular acidic pH did not activate wild type TREK2 in the presence of DTNB. (B) An intracellular acidic pH activated the chimera [TREK2 (1-353)/TASK3C] regardless of the presence of DTNB.

Fig. 5

Summary of the effects of DTNB on wild type TREK2 and mutants. (A) The amino acid sequence of the C-terminus of TREK2. (B) Membrane topologies of TREK2 and mutants show two pore-forming domains and four transmembrane segments (The membrane topology has been modified from Kim et al, 2001b). The amino acid positions indicate where the C-terminal replacements were made. The portion of the TREK2 C-terminus that was replaced with the C-terminus of TASK3. (C) The bar graph shows a summary of the effects of DTNB on wild type TREK2 and mutants.