Cryo-EM structures of Arabidopsis CNGC1 and CNGC5 reveal molecular mechanisms underlying gating and calcium selectivity

Abstract

Plant cyclic nucleotide-gated channels (CNGCs) belong to the cyclic nucleotide-binding domain (CNBD) channel family, but are phylogenetically classified in a distinct branch. In contrast to their animal counterparts of K+-selective or non-selective cation channels, plant CNGCs mainly mediate Ca2+ influx and are involved in various physiological processes, such as stomatal movements, pollen-tube growth and immune responses. Here, we present the cryo-EM structure and electrophysiological analysis of plant CNGC representatives, Arabidopsis CNGC1 and CNGC5. We found that CNGC1 and CNGC5 contain a unique extracellular domain featuring disulfide bonds that is essential for channel gating via coupling of the voltage-sensing domain with the pore domain. The pore domain selectivity filter possesses a Gln residue at the constriction site that determines the Ca2+ selectivity. Replacement of this Gln with Glu, typically observed in CNBD-type non-selective cation channels, could convert CNGC1 and CNGC5 from Ca2+-selective channels to non-selective cation channels permeable to Ca2+, Na+ or K+. In addition, we found that the CNGC1 and CNGC5 CNBD homology domain contains intrinsic-ligand-like interactions, which may devoid the binding of cyclic nucleotides and lead to gating independent of cAMP or cGMP. This research not only provides a mechanistic understanding of plant CNGCs’ function, but also adds to the comprehensive knowledge of the CNBD channels. Using cryo-EM structures and electrophysiological analysis of Arabidopsis CNGC1 and CNGC5, this study characterizes plant CNGCs as a class of CNBD channels that feature Ca2+ selectivity and are not regulated by cyclic nucleotide monophosphate binding.