3rd Edition Of Plant Science and Molecular Biology World Conference 2026

Abstract

The outward-rectifying potassium channel GORK in Arabidopsis thaliana plays a pivotal role in stomatal closure by mediating K⁺ efflux from guard cells, thereby regulating turgor pressure. However, the structural basis for the regulatory control of GORK’s gating has remained obscure. Here, we report high‐resolution cryo‐EM structures (3.16–3.27 Å) of GORK in five distinct conformational states, revealing key rearrangements in its cytoplasmic domains between the cyclic nucleotide binding domain (CNBD) and ankyrin repeat (ANK) domain, called "CNBD-Ankyrin bridge". These structures show that the CNBD-Ankyrin bridge undergoes secondary‐structure transitions, forming an α-helix in the “pre-open” state but adopting non-helical conformations in more distal closed states. The C-linker motions modulate the interactions between cytosolic domains and transmembrane voltage sensor elements (S4/S5), thereby determining whether closed conformations are closer to pre‐open or deeply closed states. Functional validation by glycine scanning and mutagenesis of key residues (residues D511-D519, H523 etc.) reveals that the structure of the CNBD-Ankyrin bridge is critical for K⁺ transport activity. These findings support a multi-step gating mechanism, in which dynamic cytosolic rearrangements act as regulatory inputs. Our results provide new mechanistic insight into how cytosolic signals may regulate GORK activity during stomatal closure, and identify potential targets for engineering stomata to optimize water use efficiency and stress tolerance in plants.