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Published on: November 11, 2016
Norfluoxetine inhibits TREK-2 K2P channels by multiple mechanisms including state-independent effects on the
Peter Proks1,2, Marcus Schewe3, Linus J Conrad1,2
1Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK.
Abstract:
The TREK subfamily of two-pore domain K+ (K2P) channels are inhibited by fluoxetine and its metabolite, norfluoxetine (NFx). Although not the principal targets of this antidepressant, TREK channel inhibition by NFx has provided important insights into the conformational changes associated with channel gating and highlighted the role of the selectivity filter in this process. However, despite the availability of TREK-2 crystal structures with NFx bound, the precise mechanisms underlying NFx inhibition remain elusive. NFx has previously been proposed to be a state-dependent inhibitor, but its binding site suggests many possible ways in which this positively charged drug might inhibit channel activity. Here we show that NFx exerts multiple effects on single-channel behavior that influence both the open and closed states of the channel and that the channel can become highly activated by 2-APB while remaining in the down conformation. We also show that the inhibitory effects of NFx are unrelated to its positive charge but can be influenced by agonists which alter filter stability, such as ML335, as well as by an intrinsic voltage-dependent gating process within the filter. NFx therefore not only inhibits channel activity by altering the equilibrium between up and down conformations but also can directly influence filter gating. These results provide further insight into the complex allosteric mechanisms that modulate filter gating in TREK K2P channels and highlight the different ways in which filter gating can be regulated to permit polymodal regulation.
Insights
Norfluoxetine (NFx) affects both open and closed states of TREK K2P channels, influencing gating via multiple mechanisms unrelated to its charge. This reveals complex allosteric regulation of channel activity.
Area of Science:
- Neuroscience
- Molecular Biology
- Biophysics
Background:
- TREK subfamily of two-pore domain K+ (K2P) channels are modulated by antidepressants like fluoxetine.
- Norfluoxetine (NFx), a metabolite of fluoxetine, inhibits TREK channels, offering insights into channel gating and selectivity filter function.
- The exact mechanisms of NFx inhibition and its interaction with TREK channel conformations remain unclear.
Purpose of the Study:
- To elucidate the precise mechanisms by which NFx inhibits TREK K2P channels.
- To investigate the influence of NFx on channel gating, including open and closed states.
- To determine the role of NFx's charge and interactions with agonists/voltage on channel activity.
Main Methods:
- Single-channel electrophysiology to analyze TREK channel behavior.
- Utilizing activators like 2-APB and ML335 to probe channel states.
- Investigating the impact of NFx charge and voltage-dependent gating on filter function.
Main Results:
- NFx affects both open and closed states of TREK channels, demonstrating state-dependent inhibition.
- Channel activation by 2-APB can occur while the channel remains in a 'down' conformation.
- NFx inhibition is independent of its positive charge but influenced by agonists and intrinsic voltage-dependent filter gating.
- NFx modulates the equilibrium between channel conformations and directly impacts filter gating.
Conclusions:
- NFx exerts complex, multimodal regulation on TREK K2P channel gating.
- Inhibition mechanisms involve altering conformational equilibria and directly influencing filter gating.
- These findings deepen the understanding of allosteric modulation in TREK channels and their polymodal regulation.
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