Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.754813
Title: Pharmacological regulation of TREK1, TREK2 and TRESK two pore domain potassium channels
Author: Walsh, Yvonne
ISNI:       0000 0004 7427 8304
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2017
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Abstract:
Introduction: Two pore domain potassium (K2P) channels are responsible for background currents that regulate membrane potential and neuronal excitability. Compounds which alter the activity of these channels are predicted to have therapeutic potential in treating CNS disorders. Members of the TREK family of K2P channels (TREK1 and TREK2) have been shown to play an active role in neuroprotection, depression and pain, whilst TRESK, with high expression in sensory neurons, has a role in nociception. Sipatrigine, a neuroprotective agent and a derivative of the anticonvulsant lamotrigine, is a known antagonist of TREK channels whilst lamotrigine is thought to primarily inhibit TRESK channels. A new compound, Cen-092-C, has also been developed which is structurally similar to lamotrigine. However, its effects on K2P channels are unknown. To understand the mechanism of channel inhibition by drugs, the structure of TREK2 was solved and was co-crystallised with norfluoxetine. This showed that fenestration sites were important in channel and current inhibition. Furthermore, TRESK docking studies showed that F145 and F352 function in a similar way to TREK2 fenestration site, as the bulky phenylalanine faces into the pore, and are thought to be important for compound binding. The aim of this study is to clarify to differences in the inhibitory effect of these compounds on the selected K2P channels and to investigate the mechanism by which these compounds inhibit the channels current. Methods: Wild-type (WT) and mutated human K2P channels were transiently expressed in tsA-201 cells. The currents were measured using whole-cell patch-clamp electrophysiology. Results: Sipatrigine was shown to inhibit both TREK1 and TREK2 current. Lamotrigine was also found to inhibit TREK1 and to a lesser extent TREK2. Cen-092-C was found to be less effective on TREK1 and TRESK current compared to sipatrigine, but similar to lamotrigine results. The sipatrigine inhibitory effect, but not lamotrigine, was reduced by mutations on the M4 region at the fenestration site of TREK1 and TREK2 (L286 and L320). This sensitivity is selective at this site as other mutations in the central cavity showed no change in sipatrigine inhibition. Interestingly, the gain-of-function mutation (TREK1 E306A) on the C terminus showed a reduced sipatrigine inhibition. The effect of sipatrigine on TREK2 showed an over-recovery of current following wash-off of the compound. The wash-off current increase was not seen if the N-terminus length is forced into intermediate and short isoform. Sipatrigine inhibition was significantly decreased when the N-terminus was truncated. Sipatrigine has been shown to strongly inhibit TRESK. Lamotrigine was seen to inhibit TRESK current, however significantly less effective compared to sipatrigine. Furthermore, lamotrigine did show state dependent inhibition when TRESK is in the fixed activated state. Cen-092-C was also found to inhibit TRESK to a similar degree to lamotrigine, however there was no state dependent inhibition on TRESK current. The effects of these antagonists on TRESK has been shown to be abolished by mutations on two sites at the central cavity (F145 and F352). Conclusion: Lamotrigine was found not to be TRESK selective, contrary to other studies. Sipatrigine and lamotrigine inhibition works through binding to the channel. The fenestration site in both TREK1 and TREK2 has been found to be an important binding site of sipatrigine, differing from lamotrigine. This suggests that the structurally similar compounds bind to different regions of the TREK channels. Furthermore, the over recovery of TREK2 current after sipatrigine wash off is believed to show the compound's biphasic effect, where the underlying enhancement of current is hidden by the action of inhibition. The N-terminus is therefore believed to be important in regulating sipatrigine action on TREK2. It remains unclear whether the TRESK potential binding sites (F1452 and F352) are important in compound binding as the inserted mutation is believed to shift the channel to constant active state. The newly developed compound Cen-092-C shows a significantly greater degree of inhibition of TRESK when compared to TREK1. Cen-092-C and lamotrigine inhibition of TRESK is not significantly different. Lamotrigine inhibition of TRESK current is state dependent whereas sipatrigine and Cen-092-C inhibition of TRESK current is shown as state independent. All of this together could lead to a better understanding of how neuroprotective agents effect TREK and TRESK channels and could contribute to the design of more efficient ligands.
Supervisor: Mathie, Alistair Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.754813  DOI: Not available
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