Cryo-EM structure of the hyperpolarization-activated inwardly rectifying potassium channel KAT1 from Arabidopsis

Abstract

Plants utilize K + ions to maintain hydrostatic pressure, drive irreversible cell expansion for growth, and facilitate reversible changes in guard cell volume that cause stomatal opening or closing.KAT1 is a voltage-dependent potassium channel from Arabidopsis thaliana that is mainly expressed in guard cells.2][3] To understand the gating mechanism of plant K + channels poses several challenges, despite many structural similarities between these plant K + channels and mammalian Kv and Shaker channels. 4emarkably, most voltage-gated ion channels, such as Na + (Nav), Ca 2+ (Cav), and K + (Kv) channels, open when the cell membrane is depolarized (when the voltage is positive inside relative to outside).Comparing with conventional depolarized K + channels, KAT1 has a uniquely reversed voltage dependence: depolarization causes closing, and hyperpolarization causes opening. 3KAT1 thus falls into a rare class of hyperpolarization-activated channels, which include hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels in animals, and KAT and AKT channels in plants.Mechanistic studies have focused predominantly on the depolarization-activated ion channels.The mechanism underlying the voltage sensor control of the gate in hyperpolarizationactivated ion channels is little studied.To date, the only resolved structure of a hyperpolarization-activated channel is that of HCN1. 5,6The cryo-EM structure of the HCN1 channel in a "hyperpolarized" state reveals that the long S4 helix breaks into two helices, with one running parallel to the membrane surface, analogous to the S4-S5 linker of domain-swapped voltage-gated channels.These findings suggest a basis for allosteric communication between voltage sensors and the gate in hyperpolarization-gated ion channels.However, preliminary sequence analysis shows that KAT1 and HCN1 have low sequence similarity.The S4 helix of KAT1 is much shorter than that of HCN1.This leads to the question of whether the structure and proposed gating mechanism of HCN1 can fully recapitulate those of KAT1.The intriguing biophysical properties of KAT1 motivate us to elucidate its molecular architecture.Full-length KAT1 from Arabidopsis was cloned and transfected into Sf9 cells for expression.However, we failed to obtain KAT1 protein from the membrane fraction.Then we co-expressed KAT1 with KAB1, a structural component of some plant K + channels, 7 and eventually obtained highly stable and homogeneous KAT1 proteins uniform in composition as indicated by gel filtration and SDS-PAGE analysis (Supplementary information, Fig. S1).Unexpectedly, the corresponding band of KAB1 was not observed in the purification gel.This indicates that KAB1 could not form complex with KAT1 in vitro, but rather acts as a chaperon that facilitates KAT1 translocation to the membrane in Sf9 cells.The purified KAT1 was subjected to cryo-EM studies.A three-dimensional EM map was reconstructed to an overall resolution of 3.2 Å (Supplementary information, Figs.S2 andS3).The secondary-structure features of

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