Coupling of two functional Gates in an Alkaline-Activated Two-Pore Domain K+ Channel

Within this work the gating structures in the TALK-2 K2P are investigated, revealing a second, inner (intracellular) permeation gate, besides the already known selectivity filter (SF) gate. Utilizing pharmacological tools, mutagenesis assays and electrophysiological methods reveal amino acid residues in transmembrane helices 2 and 4 involved in the formation of the inner gate.
Pore blocking experiments, as well as cysteine modification assays uncover means to open this inner gate in TALK-2 K2P channels. Furthermore, they reveal a strong positive coupling between the SF and the inner gate. Stimuli that activate the filter gate simultaneously open the inner gate in TALK-2. Moreover, analysis of TALK-2 tail currents uncovers reciprocal positive coupling between the two gates, resulting in a characteristic tail current cross-over of deactivating tail currents for WT channels with and without an open pore blocker bound to the inner cavity.
Finally, measurements of G-V relationships demonstrate means, like activation, to reduce the ‘mechanical load’ in TALK-2 K2P channels, describing the electrical energy required for opening TALK-2 channels.
These findings ultimately lead to the conclusion that K2P channels can be divided into “only SF gated” and “gated by the SF and an inner gate”. Moreover, the strong positive coupling between the two gates in TALK-2 appears to be a highly effective way of significantly increasing a very low basal channel activity by a wide range of stimuli. This concept of positive gate coupling could also apply to other K+ channels with two regulatory gates.


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