Structural Basis for pH-gating of the K+ channel TWIK1 at the selectivity filter.

TWIK1 (K2P1.1, KCNK1) is a widely expressed pH-gated two-pore domain K+ channel (K2P) that contributes to cardiac rhythm generation and insulin release from pancreatic beta cells. TWIK1 displays unique properties among K2Ps including low basal activity and inhibition by extracellular protons through incompletely understood mechanisms. Here, we present cryo-EM structures of TWIK1 in lipid nanodiscs at high and low pH that reveal a previously undescribed gating mechanism at the K+ selectivity filter. At high pH, TWIK1 adopts an open conformation. At low pH, protonation of an extracellular histidine results in a cascade of conformational changes that close the channel by sealing the top of the selectivity filter, displacing the helical cap to block extracellular ion access pathways, and opening gaps for lipid block of the intracellular cavity. These data provide a mechanistic understanding for extracellular pH-gating of TWIK1 and illustrate how diverse mechanisms have evolved to gate the selectivity filter of K+ channels.

High-performance microbial opsins for spatially and temporally precise perturbations of large neuronal networks.

The biophysical properties of existing optogenetic tools constrain the scale, speed, and fidelity of precise optogenetic control. Here, we use structure-guided mutagenesis to engineer opsins that exhibit very high potency while retaining fast kinetics. These new opsins enable large-scale, temporally and spatially precise control of population neural activity. We extensively benchmark these new opsins against existing optogenetic tools and provide a detailed biophysical characterization of a diverse family of opsins under two-photon illumination. This establishes a resource for matching the optimal opsin to the goals and constraints of patterned optogenetics experiments. Finally, by combining these new opsins with optimized procedures for holographic photostimulation, we demonstrate the simultaneous coactivation of several hundred spatially defined neurons with a single hologram and nearly double that number by temporally interleaving holograms at fast rates. These newly engineered opsins substantially extend the capabilities of patterned illumination optogenetic paradigms for addressing neural circuits and behavior.