Effects of divalent cations and spermine on the K⁺ channel TASK‐3 and on the outward current in thalamic neurons

Abstract

The potassium channels TASK-1 and TASK-3 show high sequence homology but differ in their sensitivity to extracellular divalent cations. Heterologous expression in HEK293 cells showed that the single-channel conductance of TASK-3 increased approximately four-fold after removal of external divalent cations, whereas the conductance of TASK-1 was unaffected. Replacing the glutamate at position 70 of TASK-3 by a lysine or arginine residue abolished the sensitivity to divalent cations. The reverse mutation in TASK-1 (K70E) induced sensitivity to divalent cations. The organic polycations spermine and ruthenium red modulated the conductance of TASK-3 in a similar way as Ca 2+ or Mg 2+ . Our data suggest that these effects were mediated by shielding of the negative charges in the extracellular loops of TASK-3. Whole-cell currents carried by TASK-3 channels were inhibited by spermine and ruthenium red even in the presence of external divalent cations. These data suggest that, in addition to their effect on single-channel conductance, spermine and ruthenium red decreased the open probability of TASK-3 channels, probably by binding to residue E70. The standing outward current in thalamocortical relay neurons, which is largely carried by TASK channels, was also inhibited by divalent cations and spermine. Using the differential sensitivity of TASK-1 and TASK-3 to divalent cations and spermine we found that about 20% of the standing outward current in thalamocortical relay neurons flows through TASK-3 channels. We conclude from our results that inhibition of TASK-3 channels may contribute to the neuromodulatory effect of spermine released from neurons during repetitive activity or during hypoxia.

An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH*

“…Independent evidence for the importance of electrostatics of the EIP comes from experiments with TASK-3 blockers Zn 2ϩ and RR. The inhibition by these, and other divalent cations, has been shown to depend on the presence of the Glu-70 residue (32,(37)(38)(39) and a neutral His-98 (32,38). Here we show that inhibition of TASK-3 by Zn 2ϩ , just as pH o gating, is cooperative and strongly impeded by increasing extracellular K ϩ concentration.…”

“…The results suggest that the main effect of Glu-70 on Zn 2ϩ inhibition is in electrostatically affecting its concentration in the EIP. A similar role for Glu-70 was surmised from the effect of divalent cations on TASK-3 single channel conductance (37). Our experiments comparing Zn 2ϩ inhibition of TASK-3 and TASK-3-E70K allowed us an estimation of the electrostatic potential due to the charge replacement at a site of action of the divalent cation.…”

Gating, Regulation, and Structure in K2P K+ Channels: In Varietate Concordia?

“…Glu70 affects the electrostatic potential in the extracellular ion pathway and might alter the occupancy of the outermost SF K 1 binding sites . The importance of electrostatics in the extracellular ion pathways is also highlighted by the effect of TASK-3 blockers Zn 21 and ruthenium red, which requires the presence of the Glu70 residue (Czirják and Enyedi, 2003;Clarke et al, 2004Clarke et al, , 2008Musset et al, 2006) and a neutral His98 (Clarke et al, 2004(Clarke et al, , 2008. Additionally, TASK-3 blockade by Zn 21 and ruthenium red is cooperative and strongly impeded by increasing concentrations of extracellular K 1 .…”

Both TASK-3 and TREK-1 two-pore loop K channels are expressed in H295R cells and modulate their membrane potential and aldosterone secretion

“…A number of putative K2P channel modulators have been used in previous studies (11,14,17,20), although their specificity is limited. Here, we used the TREK-1 inhibitors fluoxetine (10 M) and fluspirilene (1 M), as well as the TASK-3 inhibitors ruthenium red (10 M) and spermine (200 M), to investigate their effects on membrane potential of H295R cells.…”