Intrinsic Capacity, Genetic Susceptibility and Incident Cardiovascular Disease Among Individuals With Diabetes: A Prospective Cohort Study.

K potassium channels produce leak currents that are important for controlling resting membrane potential in diverse cell types. Fifteen human K channel subunits comprise six subfamilies (TWIK, TREK, THIK, TASK, TALK, and TRESK) that each respond to diverse sets of physiological cues including physical force, lipid modulation, and various signaling cascades. Structural studies of members from the TWIK, TREK, TALK, TASK, and THIK subfamilies reveal a shared architecture in which the two pore domains (PD1 and PD2) of an individual Ks subunit form a pseudo-tetrameric pore upon subunit dimerization. In contrast to other types of potassium channels, the selectivity filter (SF) “C-type” gate acts as the principal site of K modulation for most Ks. Despite their relatively small size (~60–70 kDa), Ks contain a wealth of binding sites for various classes of lipid and small molecule modulators whose roles in channel gating remain imperfectly understood. In particular, the potential role of lipid access through a lateral fenestration below the selectivity filter has attracted much attention, but whether this represents a mechanism for direct block of the pore remains a point of controversy, as simulations indicate that even though lipid tails can enter, they do not block the pore. K6.1 (TWIK2) belongs to the TWIK subfamily comprising K1.1 (TWIK1), K6.1 (TWIK2), and K7.1 and is found in the gastrointestinal tract, vasculature, and immune system where it is linked to vascular and pulmonary hypertension and sepsis responses. K6.1 (TWIK2) and K1.1 (TWIK1) are notable for their endolysosomal localization. Functional and cell biology studies indicate that in K6.1 (TWIK2), this intracellular distribution originates from internalization signals that favor this location and largely prevent plasma membrane expression. Interestingly, in macrophages, transit of endosomally sequestered K6.1 (TWIK2) to the plasma membrane following a signal initiated by extracellular ATP has been implicated in the process underlying NLRP3 (OD, RR and yrin domain-containing protein ) inflammasome activation. This K6.1 (TWIK2) function parallels that described for the THIK subfamily in NLRP3 activation in microglia and interleukin 1β (IL−1β) release, highlighting the importance of the link between K cellular location and function. Here, we report structural studies of human K6.1 (TWIK2) and mutants in lipid nanodisc and detergent environments. The data reveal a channel architecture that largely resembles K1.1 (TWIK1) having three notable features: an extra helix in the extracellular cap domain (the “ear helix”), an unusual conformation of a key residue in the first selectivity filter (SF1), and block by pair of intracellular lipids that engage the channel through a set of two-tailed interactions with lateral fenestrations linking the pore and bilayer. Elements from this last feature occupy the site that has been identified in K1.1 (TWIK1) and other Ks as a binding site for a single alkyl chain or detergent. Structures of a previously characterized K6.1 (TWIK2) mutant that increases the plasma membrane localization and activity yield plugged and unplugged forms that highlight the importance of lipid removal for channel function. Moreover, the structure of the R257A mutant that enhances function and affects a key lipid coordinating residue reveals a descent of the lipid plug towards the intracellular opening, highlighting the important role of Arg257 in lipid coordination. This key role for the Arg257 site is further supported by molecular dynamics studies that reveal dynamic interactions between Arg257 and the lipid plug. The proposal that the lipid plug forms a physical barrier that prevents ion passage and that this plug can be destabilized by mutations that enhance channel function suggests a working model in which the function of the TWIK subfamily depends on lipid plug removal.