Abstract
A better understanding of the gating of TREK two pore domain potassium (K2P) channels and their activation by compounds such as the negatively charged activator, flufenamic acid (FFA) is critical in the search for more potent and selective activators of these channels. Currents through wild-type and mutated human K2P channels expressed in tsA201 cells were measured using whole-cell patch-clamp recordings in the presence and absence of FFA. Mutation of the TM2.6 residue of TREK-1 to a phenylalanine (G171F) and a similar mutation of TM4.6 (A286F) substantially reduced current through TREK-1 channels. In complementary experiments, replacing the natural F residues at the equivalent position in TRESK channels, significantly enhanced current. Known, gain of function mutations of TREK-1 (G137I, Y284A) recovered current through these mutated channels. This reduction in current could be also be reversed pharmacologically, by FFA. However, an appropriate length MTS (MethaneThioSulfonate) cross-linking reagent (MTS14) restricted the activation of TREK-1_A286C channels by repeated application of FFA. This suggests that the cross-linker stabilises the channel in a conformation which blunts FFA activation. Pharmacologically reversible mutations of TREK channels will help to clarify the importance of these channels in pathophysiological conditions such as pain and depression.
Highlights
There is increasing evidence in support of a role of TREK channels in nociceptive processing, which may offer hope of new therapeutic advances in the treatment of pain (e.g.9,10)
We show that mutation of the TM2.6 of TREK-1 to a phenylalanine residue (G171F) and a similar mutation of TM4.6 (A286F) substantially reduces current through TREK-1 channels and, importantly, that this reduction in current can be reversed, pharmacologically, by flufenamic acid (FFA)
There is significant movement of both the TM2 and TM4 regions of the TRAAK channel in crystal structures of gain of function mutations, suggesting that these regions of the channel move when the channel is gated at the selectivity filter. Consistent with these observations on TRAAK channels, we have found that currents through loss of function TM4.6 TREK-1 (A286F) channels could be restored to 57.7 ± 6 pA/pF (n = 14) when the channels carried the equivalent gain of function mutation, G137I, (Fig. 7A–C,E), which is larger than WT current through TREK-1 channels
Summary
There is increasing evidence in support of a role of TREK channels in nociceptive processing, which may offer hope of new therapeutic advances in the treatment of pain (e.g.9,10) Fenamate compounds, such as flufenamic acid (FFA), are non-steroidal anti-inflammatory drugs (NSAIDs) used clinically in the treatment of pain. These compounds have been shown to up regulate the activity of TREK channels[11,12]. Mutation of this TM2.6 residue to an aspartate or asparagine in all K2P channels studied, produces a gain of function channel[18] These mutations cause substantial increases in channel open probability (Po) with no substantive change in single channel conductance or the amount of channel expressed at the cell membrane. This has been proposed to give a simple and powerful strategy to systematically manipulate activity of the entire K2P channel family[18]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
