The pore structure and gating mechanism of K2P channels.

Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K(+) channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing.

Molecular basis of inward rectification: polyamine interaction sites located by combined channel and ligand mutagenesis.

Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter.

Phosphatidylinositol 4,5-bisphosphate (PIP2) modulation of ATP and pH sensitivity in Kir channels. A tale of an active and a silent PIP2 site in the N terminus.

Phosphatidylinositol polyphosphates (PIPs) are potent modulators of Kir channels. Previous studies have implicated basic residues in the C terminus of Kir6.2 channels as interaction sites for the PIPs. Here we examined the role of the N terminus and identified an arginine (Arg-54) as a major determinant for PIP(2) modulation of ATP sensitivity in K(ATP) channels. Mutation of Arg-54 to the neutral glutamine (R54Q) and, in particular, to the negatively charged glutamate (R54E) impaired PIP(2) modulation of ATP inhibition, while mutation to lysine (R54K) had no effect. These data suggest that electrostatic interactions between PIP(2) and Arg-54 are an essential step for the modulation of ATP sensitivity. This N-terminal PIP(2) site is highly conserved in Kir channels with the exception of the pH-gated channels Kir1.1, Kir4.1, and Kir5.1 that contain a neutral residue at the corresponding positions. Introduction of an arginine at this position in Kir1.1 channels rendered the N-terminal PIP(2) site functional largely increasing the PIP(2) affinity. Moreover, Kir1.1 channels lose the ability to respond to physiological changes of the intracellular pH. These results explain the need of a silent N-terminal PIP(2) site in pH-gated channels and highlight the N terminus as an important region for PIP(2) modulation of Kir channel gating.