Ca2+-regulated Ca2+ channels with an RCK gating ring control plant symbiotic associations

Sunghoon Kim,Jun Liao,Shane Bernard,Muthusubramanian Venkateshwaran,Youxing Jiang,Jean-Michel Ane,Weizhong Zeng

doi:10.1038/s41467-019-11698-5

Abstract

A family of plant nuclear ion channels, including DMI1 (Does not Make Infections 1) and its homologs CASTOR and POLLUX, are required for the establishment of legume-microbe symbioses by generating nuclear and perinuclear Ca2+ spiking. Here we show that CASTOR from Lotus japonicus is a highly selective Ca2+ channel whose activation requires cytosolic/nucleosolic Ca2+, contrary to the previous suggestion of it being a K+ channel. Structurally, the cytosolic/nucleosolic ligand-binding soluble region of CASTOR contains two tandem RCK (Regulator of Conductance for K+) domains, and four subunits assemble into the gating ring architecture, similar to that of large conductance, Ca2+-gated K+ (BK) channels despite the lack of sequence similarity. Multiple ion binding sites are clustered at two locations within each subunit, and three of them are identified to be Ca2+ sites. Our in vitro and in vivo assays also demonstrate the importance of these gating-ring Ca2+ binding sites to the physiological function of CASTOR as well as DMI1.

Highlights

Make Infection 1 (DMI1) from M. truncatula has been recognized as a cation channel that resides in the nuclear envelope and is directly involved in generating oscillations of the nuclear and perinuclear concentration of Ca2+ often referred to as Ca2+ spiking[6,7,8,9,10]
Our initial attempt to measure the channel activity by patching the plasma membrane of human embryonic kidney (HEK) 293 cells expressing full-length LjCASTOR was not successful due to the lack of surface expression of the channel and the majority of the expressed proteins being localized in the endoplasmic reticulum (ER) and nuclear membranes
Using LjCASTOR as our model system, we demonstrated that the plant nuclear ion channels, including DMI1, POLLUX, and CASTOR, function as tetrameric Ca2+-regulated Ca2+ channels whose ligand-binding domains form an RCK gating ring

Summary

Introduction

Make Infection 1 (DMI1) from M. truncatula has been recognized as a cation channel that resides in the nuclear envelope and is directly involved in generating oscillations of the nuclear and perinuclear concentration of Ca2+ often referred to as Ca2+ spiking[6,7,8,9,10]. Sequence analysis suggested that the last two TM regions form the pore and the N-terminal half of the soluble domain is distantly homologous to a Regulator of K+ Conductance (RCK) domain[6,11,12,14]. Initial studies using yeast rescue assays and artificial lipid bilayers suggested that CASTOR, POLLUX, and DMI1 could be K+ channels[12,24] with the RCK-containing MthK channel used as a homology model for a part of these channels (the last two TM regions and the first half of the soluble domain)[11]. One current model of the early symbiotic signaling pathway speculates that DMI1, CASTOR, and POLLUX function as ligand-gated K+ channels that can modulate the membrane potential across the nuclear envelope and that they work together with CNGC channels to modulate nuclear Ca2+ release[12,25]. If the soluble domain of these channels serves as a ligand binding domain similar to other RCK-regulated channels, the functional ligand remains to be identified

Methods

Results

Conclusion