Ruthenium red inhibits TASK-3 potassium channel by interconnecting glutamate 70 of the two Subunits

TASK channels are highly pH-sensitive two-pore-domain background potassium channels expressed in the central nervous system and in some peripheral tissues. Their current can be regulated by receptor-mediated activation of phospholipase C and also by pharmacological means. We have reported previously that the cationic dye, ruthenium red (RR), inhibited homodimeric TASK-3 (kcnk9), whereas TASK-1 (kcnk3) homodimer and TASK-1/TASK-3 heterodimer were not affected by this compound. In the present study, we identify the molecular determinant of the RR-mediated TASK-3 inhibition. Mutation of the negatively charged Glu 70 of TASK-3 to Arg (E70R) or Cys (E70C) abolished the inhibitory action of RR. When two TASK-3 coding sequences were concatenated, and the entire homodimer was expressed as a single polypeptide chain, the resulting tandem channel was also sensitive to RR. Mutation of Glu 70 in either the first (E70R) or the second (E465R) linked subunit prevented the action of the inhibitor. Together with the Hill coefficient of 1.0 for TASK-3 inhibition, these data indicate that simultaneous binding of one polycationic RR molecule to Glu 70 of both subunits is required for the inhibitory action. The pivotal role of this residue in the inhibitory mechanism of RR is confirmed by the gained RR sensitivity of the mutant TASK-1 in which Lys 70 was changed to Glu. Our results indicate that RR inhibits TASK-3 by tethering its two subunits and identify amino acid 70 as a possible target for designing selective inhibitors against the different TASK channels.