Plant Cyclic Nucleotide-Gated Channels: New Insights on Their Functions and Regulation

Calcium (Ca2+) signaling is crucial for all aspects of plant physiology, including defense, abiotic stress responses, and development; recent research has elucidated the role of plant cyclic-nucleotide–gated channels (CNGCs) in Ca2+ signaling and downstream processes. CNGCs belong to the superfamily of voltage-gated ion channels. Like voltage-gated K+ channels, animal CNGCs, and hyperpolarization and cyclic nucleotide-regulated channels, plant CNGCs are tetrameric and have six transmembrane domains, with a cytosolic N-terminal (NT) and C-terminal (CT) region per subunit (Jegla et al., 2018). The first plant CNGC isoforms were identified as calmodulin (CaM)-binding proteins in 1998 (Köhler and Neuhaus, 1998; Schuurink et al., 1998); over the past five years, pioneering work has established CNGCs as Ca2+-permeable channels involved in Ca2+ oscillations and possibly receptor-mediated signaling. The spatio-temporal variation in cytosolic Ca2+ concentrations affects a wide range of cellular responses (Webb et al., 1996). For example, Ca2+ flux across the plasma membrane is an early signaling step in establishing symbiosis and immunity (Zipfel and Oldroyd, 2017). Moreover, Ca2+ affects many developmental processes: repetitive spiking or oscillations in cytosolic Ca2+ concentration entrain circadian rhythms, underlie polar expansion of root hairs and pollen tubes, occur in response to the application of auxin to elongating root cells, and control stomatal movements in response to CO2 and abscisic acid (Felle, 1988; McAinsh et al., 1995; Holdaway-Clarke et al., 1997; Allen et al., 2001; Love et al., 2004; Monshausen et al., 2008). The production of Ca2+ oscillations requires positive and negative feedback regulation, and theoretical modeling of Ca2+ oscillations in plants has been successfully applied to some model systems (Martins et al., 2013; Liu et al., 2019). However, understanding Ca2+ dynamics on the molecular and quantitative levels in plants has been hampered by lack of knowledge about the molecular nature and regulation of the channels that allow Ca2+ entry. In this Update, we summarize recent advances in physiological, biochemical, and electrophysiological characterization of CNGCs, giving new insight into the molecular functions and regulation of plant CNGCs, focusing on subunit assembly, phosphorylation, and CaM binding. Progress in understanding the assembly, activation, and regulation of plant CNGCs has been slow. This may be due in part to the pronounced differences to their animal counterparts: In contrast to early assumptions that CNGCs were non-selectively permeable to cations (Talke et al., 2003), new research shows that several CNGCs conduct Ca2+ but often do not allow K+ to cross. Table 1 summarizes our current knowledge about the regulation by cyclic nucleotide monophosphates (cNMPs) and CaM of distinct CNGC subunits expressed in heterologous expression systems, such as Xenopuslaevis oocytes and human embryonic kidney (HEK) cells. Controversial findings regarding the requirement of elevated cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP) levels indicate that our current assumptions about CNGC gating may require major revision in the near future. As an example, Arabidopsis (Arabidopsis thaliana) CNGC2 was among the first cloned family members (Köhler and Neuhaus 1998). Initial electrophysiological characterization in X. laevis oocytes suggested that CNGC2 is a voltage-dependent K+-permeable channel activated by cAMP or cGMP (Table 1; Leng et al., 1999, 2002). In comparison, CNGC4, which together with CNGC2 forms subgroup B of clade IV of Arabidopsis CNGCs (Mäser et al., 2001), was reported to encode a voltage-independent cAMP- and cGMP-gated channel (Balagué et al., 2003). Recent work suggests that at least in X. laevis oocytes, both channels work as a hyperpolarization-activated calcium-permeable channel in a heteromeric assembly, without requirement for the elevation of cNMPs levels (Tian et al., 2019). Similarly, patch-clamp recordings on plasma membranes of plant cell protoplasts have detected cNMP-dependent stimulation of hyperpolarization-activated Ca2+-permeable channels, and these could in some cases be attributed to distinct CNGC isoforms, as in case of e.g. CNGC5 and CNGC6 in Arabidopsis guard cells (Wang et al., 2013). However, depending on the expression system and experimental condition, there seems to be no absolute requirement for the elevation of cNMP levels above the resting state (Table 1), and cNMP affinities and binding dynamics have in most cases not been well studied. The usage of genetically encoded reporters for cAMP or cGMP (Isner and Maathuis, 2013; Jiang et al., 2017) and precise biochemical analysis may provide more definite answers regarding the physiological importance of cNMPs to activate CNGCs in vivo. In light of recent advances toward the characterization of phosphorylation and CaM binding as gating agents or modifiers, the regulation by cNMPs will have to be re-evaluated. cNMPs might act only on a subset of CNGC subunits, they may act as a cofactor rather than a true trigger for ligand-activation, or they may modify the voltage-dependence or affinities toward other regulators. In addition, several recent reports showed that the universal calcium sensor protein CaM plays a more complex and significant role in regulating CNGCs than previously thought (see below for details). Therefore, despite the significant progress in recent years, the permeability, functional regulation, and nature of ligands of plant CNGCs still need further studies. In animals, phosphorylation is one way to regulate CNG and HCN channels (Kaupp and Seifert, 2002; Herrmann et al., 2015). For example, the vertebrate CNGCs CNGA1 and CNGB2 function as hetero-tetrameric channels in rod photoreceptors and the phosphorylation status of Tyr residues in these channels controls their activity (Molokanova et al., 2003). Likewise, Tyr phosphorylation alters the gating of the HCN4 pacemaker channel (Li et al., 2008). Early pharmacological studies showed that protein kinase inhibitors prevent the activity of hyperpolarization-dependent calcium channels in plant cells (Köhler and Blatt 2002; Stoelzle et al., 2003), indicating that protein phosphorylation plays a critical role in stimulus-specific Ca2+ signaling. In recent years, regulation via direct phosphorylation by Ca2+-dependent protein kinases (CDPKs/CPKs), has been documented for a number of plant ion channels, including the K+ channel KAT1, SLOW ANION CHANNEL-ASSOCIATED1, and TWO-PORE CHANNEL1 (Geiger et al., 2009; Maierhofer et al., 2014; Ronzier et al., 2014; Kintzer and Stroud, 2016; Bender et al., 2018). In an extensive survey of CPK substrates in Arabidopsis, Curran et al. (2011) identified CNGC6, CNGC7, CNGC9, and CNGC18 as potential targets of CPK1, CPK10, or CPK34. So far, a specific CPK–CNGC interaction has only been shown for the kinase domain of CPK32 and CNGC18 by yeast two-hybrid assays (Y2H) and Förster resonance energy transfer analysis (Table 2; Zhou et al., 2014). Coexpression of the constitutively active form of CPK32 in X. laevis oocytes strongly enhanced CNGC18 channel activity, although actual phosphorylation was not shown and the phosphorylation sites in CNGC18 were not identified (Zhou et al., 2014). Positive regulation of CNGCs by CDPKs opens the possibility that an initial Ca2+ influx may precede activation of CNGCs by CDPKs. In this scenario, CNGCs may amplify or modify a Ca2+ response initiated by a different channel or from an internal calcium store, because some CDPK activation requires elevated Ca2+concentration. So far, negative regulation of CNGC activity by CDPKs has not been reported, but is possible. In any case, this notion supports the idea that CNGCs are part of larger protein complexes that include other channels, pumps, and decoders such as CDPKs, or are localized in proximity to these players. This notion is discussed in detail later in this article (Fig. 1) TEVC, Two-electrode voltage clamp; BiFC, bimolecular fluorescence complementation; CoIP, co-immunoprecipitation; nd, not determined. Model of a CNGC-containing signal complex (nanodomain/channelosome, shown in darker gray). A heterotetrameric CNGC channel is part of a sensing receptor complex containing PRRs, their clients (e.g. BIK1, RESPIRATORY BURST OXIDASE HOMOLOG D, RLCK), various pumps (e.g. proton ATPase and Ca2+ pumps), and decoders (e.g. CPKs and CaM). The formation of such a signal complex can be permanent or temporal upon recognition of specific stimuli (transient signaling complex) and the combination of specific players can contribute to generate precise spatiotemporal Ca2+ signals. Recruitment of CNGCs in a specific signaling complex may be achieved by MLO proteins. Phosphorylation plays significant roles to activate CNGCs or induce their turnover by E3 ubiquitin ligases and the 26s proteasome. V, Vesicle. Considering the plasma membrane localization of most plant CNGCs, receptor kinases, or receptor-like kinases (RLKs) are likely candidates for the kinases that phosphorylate CNGCs. Indeed, Ladwig et al. (2015) reported that CNGC17 binds to the Arabidopsis H+-ATPases (AHA), AHA1 and AHA2, as well as to BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). BAK1 is a Leu-rich repeat RLK (LRR-RLK), which can associate with various pattern recognition receptors (PRRs) as a coreceptor, forming functional receptor complexes that regulate a wide variety of physiological responses from growth to immunity (Kim and Wang, 2010; Ranf, 2017; Liang and Zhou, 2018). The growth-regulating phytosulfokine (PSK) receptor PSKR1, another LRR-RLK superfamily member, also binds to AHA1, AHA2, and BAK1, suggesting that CNGC17, PSKR1, BAK1, and AHAs may form a protein nanocluster to initiate downstream signals (Fig. 1; Ladwig et al., 2015). In addition, these interaction data suggest that BAK1 or other LRR-RLKs phosphorylate plant CNGCs. In 2019, three studies revealed that LRR-RLKs and related kinases phosphorylate CNGCs, and examined the physiological relevance of this phosphorylation (Tian et al., 2019; Wang et al., 2019; Yu et al., 2019): Tian et al. (2019) reported the relevance of CNGC phosphorylation in the recognition of pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) in of the cytosolic Ca2+ concentration is for the recognition of such as the or et al., 2014; et al., 2015). recognition by the kinase a receptor complex of BAK1, and KINASE1 to of these kinases and of BIK1, which downstream signaling and A downstream of is the RESPIRATORY BURST OXIDASE HOMOLOG D, which is for the immunity et al., 2014; et al., 2015). Tian et al. (2019) reported that CNGC4, but not also with BIK1, and is at the cytosolic domain upon recognition via (Fig. is a kinase that is a of complexes with such as and RECEPTOR KINASE1 and CNGC2 and and form a functional heteromeric which is in the of CaM et al., 2013; Tian et al., 2019). can activate this heteromeric channel in the of the possibly via phosphorylation of CNGC4, and was suggested to induce Ca2+ influx in response to Tian et al. (2019) showed CNGC2 as and as are positive of only specific calcium concentrations as their showed this condition, but calcium concentrations reported that is to activate However, both and are to calcium and have et al., 1998; et al., et al., Wang et al., 2017). Ca2+ stress Ca2+ levels et al., the possibility that they to many including Therefore, studies which channels the Ca2+ response Ca2+ in and the in these is a or of other In another recent Wang et al. (2019) examined the role of CNGC phosphorylation in and cell The for CNGC2 and complex and such as with elevation of acid levels and expression of but in the response et al., 1998; et al., et al., In the of Arabidopsis and also A was for the of CNGC4, et al., and the cell and which a functional (Wang et al., 2019). The cell and shows and calcium and indicating that has a significant role in (Wang et al., 2019). the receptor-like kinase with and and this phosphorylation channel function 1 and 2; Wang et al., 2019). and with the receptor RECEPTOR KINASE1 et al., Therefore, CNGCs a downstream of phosphorylation the Arabidopsis of is which has been with Ca2+ growth et al., et al., 2017; et al., et al., and not CNGC2 or will be to the functional of CNGCs in different plant In the new from 2019, Yu et al. (2019) a to an Arabidopsis CNGC as a in the of cellular which is by BAK1 and Like CNGC2 and CNGC4, the related channels and have been in immunity and et al., et al., Likewise, a role in and has been for and which in the CNGC family et al., et al., 2019). BAK1 and are involved in a wide variety of physiological and the due to of et al., et al., Yu et al. (2019) a of cell in plants and that a of this cell they showed that BAK1 the cytosolic domain of (Table sites are in which can also be by This revealed a of CNGC regulation in which phosphorylation of the and protein to their and form a hetero-tetrameric channel that cell and phosphorylation turnover to cellular (Fig. three recent studies showed that phosphorylation of CNGCs plays roles in their regulation (Table will be phosphorylation the channel or a will the or for cNMPs or of other CNGCs may patterns and will CNGC subunit regulation of activity or turnover by The of and the CaM binding suggested that these signaling regulate CNGCs et al., et al., et al., However, a domain to the which is by an and is among CNGCs et al., this Moreover, CaM binding sites were the cytosolic domain and of the and both positive and negative regulation of CNGCs by CaM was shown for (Fig. 2; et al., 2010; et al., The that the binds CaM in a and is for channel function the way for the gating of CNGCs, because CaM could as a subunit that in Ca2+ concentration upon channel and Ca2+-dependent feedback regulation et al., 2016; et al., 2017; et al., 2018). However, data are about the role of cNMPs and their with to a understanding of the gating of CNGCs. different of CaM regulation in CNGC is to the domain in the of the channel or the can be upon hyperpolarization to allow Ca2+ entry. of cytosolic of which to of as shown for and binding of in channel and indicate Ca2+ and Ca2+ CaM is shown with and with or Ca2+ For of CNGC subunits are shown for complex The of the cyclic nucleotide binding domain are by the domain in The state is by a and a of the with the transmembrane part of the in to (Li et al., 2017). In B and the is and the is from the membrane via the to the In X. laevis oocytes, CaM binding to the of and to the complex ion channel function (Tian et al., 2019; et al., is not which CaM binding is involved and this an elevation of in both of the as a negative (Fig. and but not et al., The by which their regulation because all Arabidopsis CaM isoforms with the of and CNGC6 in assays et al., 2017). The and isoforms have protein for one to the by et al. CaM is across which about the expression levels and physiological relevance of the by et al. the negative regulation of CNGCs on of the this will in the Ca2+ in vivo. In the case of this will to Ca2+ oscillations in root hairs and growth et al., et al., et al., 2019). In Ca2+-dependent binding of CaM to the binding of channel activity et al., expression of with a which constitutively to the by a constitutively active Therefore, CaM may regulate channel activity via binding to the and As channel activity is on heteromeric subunit et al., 2019; Tian et al., one in Ca2+ signaling research is the of channel including their CaM three the and the with and the with et al., A in a that been shown to be crucial for CaM binding et al., in a of indicating CaM binding to this CNGC function et al., 2010; et al., of the to the interaction with CaM and channel a channel of the of and the of is expressed in is by constitutively activated Ca2+ flux et al., et al., 2019). expression of the not induce et al., only the Ca2+-dependent interaction of CaM with the domain was channel function could be suggesting that the CaM complex supports channel function et al., 2017). This was further by heterologous expression of and in X. laevis hyperpolarization-dependent Ca2+ were enhanced by upon with or which was in the state by all sites et al., 2019). In comparison, was as a channel in X. laevis oocytes, both in the and of CaM et al., 2019). which is to was to activate in X. laevis oocytes, but was not et al., 2019). This to CaM despite the of and to to the as well as to the domain of in yeast et al., 2017). The and protein and in five with this to such differences in protein the functional interaction of to CNGCs suggest the that CaM may function as a Ca2+ sensor of CNGCs. CaM has and with by a Ca2+ with different which to the of CaM to regulate many proteins et al., 2014). can with the domain of CNGCs via indicating that to CNGCs in the resting state and plays a role as a subunit for the channel complex et al., 2017). Indeed, is for Ca2+ sensing and for channel at least in some channels such as et al., 2016; et al., 2017; et al., and (Fig. 2; et al., the complex may Ca2+ oscillations pollen As both and of the critical roles in CaM et al., the that some CNGCs are activated by CaM by binding of to their are new about the of the interaction of CaM with different CNGC subunits and heteromeric CNGC complexes (Fig. Therefore, more quantitative and of CaM with CNGCs are to our understanding of any gating recent have light on the role of CNGCs as of plant Ca2+ This is not because their CNGCs function as (Fig. In pollen tubes, CNGC18 is for and growth et al., et al., and have functions in pollen growth et al., 2013). In different heterologous expression systems, CNGC18 hyperpolarization-activated calcium but the regulation of channel to be complex (Table In cells, of cAMP and cGMP activated CNGC7, and CNGC18 to calcium at et al., In another CNGC18 expressed in X. laevis oocytes could be activated by of a constitutively active form of the Ca2+-dependent protein kinase CPK32 (Zhou et al., 2014). The suggested that Ca2+-dependent stimulation of calcium via CPK32 Ca2+ oscillations in pollen (Zhou et al., 2014). In later CNGC18 was active in X. laevis oocytes in the of plant kinases and without of cyclic et al., to the of CNGC18 regulation by CPK32 and cyclic in pollen A recent a for regulation by heteromeric channel and CaM in the of elevated levels of cyclic (Table 1; et al., 2019). In X. laevis oocytes, CNGC18 were by of or and this was in the of and channels in the and of studies revealed that the CNGC with other and with or Ca2+ of the for the and CNGC18 from to suggesting that Ca2+ the of from the heteromeric channel which to channel In this scenario, the heteromeric complex be active at is but a in trigger CaM and channel et al. (2019) suggest a new model in which the of of the channel complex (Fig. This of Ca2+ feedback regulation the theoretical for the in pollen However, no calcium current membrane was in oocytes, the been the of Ca2+ concentrations of current in the of with were only than with The by et al. (2019) new and data for modeling of the Ca2+ the suggested can be in vivo. of Ca2+ oscillations could also knowledge about by membrane as well as and of CaM binding. of root including and as well as of the root et al., 2017; et al., indicating role in the regulation of cell polar CNGC6, and also contribute to the of cell expansion and of cytosolic Ca2+ oscillations et al., 2019; et al., of the by the calcium oscillations and growth The of in root hairs was not established in the and et al., 2019). However, this was still in and although with as the major pacemaker in vivo. the experimental the initiated root which to the growth In another growth of the could be by of of the CNGC subunits, indicating functions of these channels et al., to the for heterologous expression of or hyperpolarization-activated Ca2+ in cells, although the role of cyclic for channel activation studies (Table 1; et al., 2016; et al., In to cytosolic Ca2+ the of CNGCs in Ca2+ oscillations has also been reported et al., 2016; et al., 2019). from and Arabidopsis are the only CNGCs that are localized to the they in Ca2+ which are crucial for root growth and symbiosis et al., 2016; et al., 2019). Ca2+ signals in many physiological one is specific stimuli generate signals to signaling The of Ca2+ and Ca2+ and binding to and the the of the McAinsh and of plant CNGCs a to generate patterns of Ca2+ on the is to that subunit has a of regulation by phosphorylation and CaM but also a of functional The Arabidopsis CNGC family has members into five (Mäser et al., have family such as or but other have many different channel subunits, such as or to the of a et al., 2015). or subunit have been or suggested for and et al., 2013; et al., 2019; et al., 2019; Tian et al., 2019). et al. (2019) reported an of or on CNGC18 heteromeric channel indicating that some CNGC members may or modify the activity of their heteromeric channel complexes (Fig. 2; Table This that the of has a on channel function and regulation, their physiological for Ca2+ is the formation of protein complexes at the plasma membrane as specific sensing (Fig. This idea is by the of of CNGCs with receptor kinases and other proteins as discussed above (Table 2; Ladwig et al., Wang et al., 2019; Yu et al., 2019; et al., the membrane by and of plasma membrane proteins in or may signaling that in plant signaling et al., 2010; et al., 2013; and For example, the BAK1 is also a of the major receptor sensing forms an active receptor complex with BAK1, signaling (Kim and Wang, Wang et al. (2015) showed that to membrane and that this of is for signal et al. showed that and to distinct plasma membrane and such spatiotemporal of receptor kinases could contribute to their signaling in immunity and growth is to that CNGC hetero-tetrameric channels are of sensing together with specific receptors and downstream to specific downstream (Fig. As discussed recent studies have the of CNGC research (see As these new data and further for our understanding of this channel and role in plant calcium signaling (see The interaction of CNGCs with receptor-like kinases and other as reported for proteins et al., allow specific CNGCs together with to be part of different with their membrane may include proteins such as The of such generate stimulus-specific Ca2+ that are by the proteins will be an for CNGC research (Fig. modeling animal has our understanding et al., et al., et al., but the of the CaM binding Therefore, of plant CNGCs will our understanding of their gating and regulation The first CNGC family was identified in 1998 in a for CaM binding targets in a from cells, and this CNGC was CaM et al., 1998). the Arabidopsis to animal CNGCs were identified and and CNGC2 (Köhler and Neuhaus, 1998). The CNGC was for family the of et al. Indeed, the of a cyclic domain the most this domain binding affinities of cAMP or cGMP to this have not been and the role of these both for channel and for physiological is still there is a about the production of cNMP in plants et al., 2010; In light of recent advances in understanding CNGC assembly, and regulation by is to conduct more quantitative by reporters for as well as biochemical and to and gating and of membrane containing CNGCs. will provide with a understanding of the role of cNMP for CNGC In this Update, we new findings on the molecular functions of plant CNGCs and discussed their this we are a new of research on CNGCs and calcium and we that more advances in this research will in the near future.

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