Functional and structural characterization of gating mechanisms in K2P channels

The regulation of electrical stimuli is a key element of cellular communication within all living organisms. Centre of this regulation are membrane spanning ion channels, that allow ion currents essential for neuronal activity and electrical signal propagation through an organism. The mechanisms that underlie the opening or closing of their selective pores (gating) is very diverse and differs among the distinct ion channel families. This work investigates the gating control of potassium channels of the tandem of two-pore-domain (K2P) channel family with a special focus on the relation of structural properties and the regulation of channel activity by pharmacological and physiological stimuli. By screening 12 of the 15 mammalian K2P channels, this work reveals that most K2P channels are regulated by negatively charged lipids, such as PIP2 and Oleoyl-CoA. The response to those lipids, however, varied strongly within the K2P channel family. However, with few exceptions, similar effects were observed for members of the individual subgroups, with TREK, TALK and THIK channels being activated and TWIK, TASK and TRESK channels being inhibited by either one or both tested lipids. In addition, this study provides evidence, that PIP2 stabilizes the closed conformation of an intracellular gate found in TASK-1 and TASK-3 channels. To study the relation of these gating processes with the structural properties of the investigated K2P channels compounds such as open channel blocker (i.e. TPA, A1899, AVE0118) as well as negatively charged activators (i.e. BL-1249, ML67-33, DCPIB) or registered drugs such as ONO-RS-082, 2-APB or Norfluoxetine were screened within this work. Additionally, mutagenetic analyses of gating critical residues as well as cysteine modifying MTS-reagents were deployed to study the involvement of the C-terminal domain in channel gating and to investigate the intracellular pore accessibility of K2P channels. As a result, this work provides further evidence that TREK-1 and TRAAK channels are activated by the engagement of the C-terminal domain with the lipid bilayer. In addition, it is shown that TALK-2 and TRESK channels comprise a state dependent pore accessibility, suggesting that these channels possess a lower cytoplasmatic gate, similar to the recently discovered intracellular gates in TASK-1 and TASK-2 channels. Finally, this work presents a novel classification of K2P channels upon their gating regulation. It includes K2P channels possessing a lower intracellular gate and an upper voltage-gate at the selectivity filter (i.e. TASK-1, TASK-2, TASK-3, TALK-2, TRESK) and K2P channels that are strictly gated at the selectivity filter (i.e. TREK-1, TREK-2, TRAAK and TWIK-1).



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