The crystal structure of TRAAK has an open inner helical gate (). The central cavity under the selectivity filter is wide and forms a vestibule continuous with the cytoplasmic solution presenting no steric hindrance to ion flux. The pore domain 1 inner helices are wide open with the smallest constriction measuring ~12 Å, similar to the most open configuration observed in MthK (fig. S10
). The pore domain 2 inner helices are slightly less open, with the smallest constriction measuring ~10 Å, but this is still more open than observed in open-state Kv structures (fig. S10
It is interesting to speculate on the potential for inner helix gating in TRAAK. Recent studies have suggested that the closely related K2P TREK-1 has an inner gate that is constitutively open (31
). Three features of the TRAAK pore domain 1 inner helix may be relevant if TRAAK has a similarly constitutively open inner gate: the presence of P155 that kinks the helix, the nature of the putative membrane interaction region (discussed below), and the peptide linkage to the pore domain 2 outer helix. The combination of these structural features may restrict the ability of the pore domain 1 inner helix to access a closed inner gate conformation.
TRAAK (and TREK) channels are exquisitely responsive to the chemical and mechanical properties of the lipid bilayer. Chemically, TRAAK is activated by arachidonic acid, other polyunsaturated fatty acids, and lysophosphatidic acidby an apparently direct mechanism (16
). Mechanically, applying the equivalent to positive (and not negative) intracellular pressure through a patch pipette reversibly activates TRAAK and TREK (21
), while hyperosmolarity-induced cell shrinkage reduces TREK currents (20
). Pressure-induced activation of TRAAK is enhanced upon cytoskeletal disruption or patch excision suggesting that TRAAK directly responds to the physical state of the bilayer (21
What might be the molecular sensor(s) of the mechanical and chemical state of the bilayer in TRAAK? In TREK channels, a region immediately C-terminal to the intracellular end of the pore domain 2 inner helix has been demonstrated to be involved in activity modulation by mechanical force, lipids and fatty acids, acidic pHi
, phosphorylation, and interaction with the accessory protein AKAP150 (20
). The homologous C-terminal region in TRAAK is included in the crystal construct and is partially modeled as a cytoplasmic extension of the pore domain 2 inner helix (). However, this region has not been shown to be involved in TRAAK modulation: TRAAK is not activated by acidic pHi
, TRAAK does not interact with AKAP150, and chimeras exchanging this region with that from TASK K2P channels display unaltered response to arachidonic acid and mechanical force (34
). Together these data suggest that an alternative molecular sensor is utilized for TRAAK lipid- and mechano-activation.
A distinctive structural feature of TRAAK is illustrated in . After the kink at P155, the pore domain 1 inner helix projects laterally and runs approximately parallel to the cytoplasmic membrane surface. Along this projection the helix is amphipathic. Hydrophobic residues point towards the lipid bilayer along one face opposite a series of basic residues (R167, R173, H174, H178) pointed towards the membrane/cytoplasm interface. An additional basic residue (K185) further along the helix is also directed towards the same plane. These five residues are conserved in basic character among TRAAK channels (fig. S3
). The amphipathic helix is thus positioned to interact with both the hydrophobic tails and acidic headgroups of membrane lipids. This structured extension of the pore domain 1 inner helix, which extends like a tendril out into the lipid membrane inner leaflet, may be related to this channel’s ability to respond to both mechanical and chemical properties of the cell membrane.
TRAAK channel inner helices and gating implications
Another consequence of the inner helix structure in TRAAK is illustrated in . The relative orientation of inner helices creates an extended lateral opening to the central cavity from the membrane. Between the two protomers, a gap ~5 Å wide extends through the bilayer from the bottom of the selectivity filter to the ends of the pore domain 2 inner helix and pore domain 1 outer helix on the cytoplasmic side. While we observe electron density from the central cavity to the lateral openings in TRAAK, we are not able to confidently assign and model a ligand at this resolution. Lateral openings have been observed in other ion channels, including the K+
channel KcsA (27
) and the voltage-gated sodium channel (42
), but the size and shape of the openings in TRAAK are striking. This bilayer-facing opening results in a large protein surface accessible to the membrane and is a potential site for the interaction of lipids and other hydrophobic molecules with the TRAAK channel.