AtKC1 is a general modulator of Arabidopsis inward Shaker channel activity

A functional Shaker potassium channel requires assembly of four α-subunits encoded by a single gene or various genes from the Shaker family. In Arabidopsis thaliana, AtKC1, a Shaker α-subunit that is silent when expressed alone, has been shown to regulate the activity of AKT1 by forming heteromeric AtKC1-AKT1 channels. Here, we investigated whether AtKC1 is a general regulator of channel activity. Co-expression in Xenopus oocytes of a dominant negative (pore-mutated) AtKC1 subunit with the inward Shaker channel subunits KAT1, KAT2 or AKT2, or the outward subunits SKOR or GORK, revealed that the three inward subunits functionally interact with AtKC1 while the outward ones cannot. Localization experiments in plant protoplasts showed that KAT2 was able to re-locate AtKC1 fused to GFP from endomembranes to the plasma membrane, indicating that heteromeric AtKC1-KAT2 channels are efficiently targeted to the plasma membrane. Functional properties of heteromeric channels involving AtKC1 and KAT1, KAT2 or AKT2 were analysed by voltage clamp after co-expression of the respective subunits in Xenopus oocytes. AtKC1 behaved as a regulatory subunit within the heterotetrameric channel, reducing the macroscopic conductance and negatively shifting the channel activation potential. Expression studies showed that AtKC1 and its identified Shaker partners have overlapping expression patterns, supporting the hypothesis of a general regulation of inward channel activity by AtKC1 in planta. Lastly, AtKC1 disruption appeared to reduce plant biomass production, showing that AtKC1-mediated channel activity regulation is required for normal plant growth.     

The Role of the C-Terminus for Functional Heteromerization of the Plant Channel KDC1

Voltage-gated potassium channels are formed by the assembly of four identical (homotetramer) or different (heterotetramer) subunits. Tetramerization of plant potassium channels involves the C-terminus of the protein. We investigated the role of the C-terminus of KDC1, a Shaker-like inward-rectifying K⁺ channel that does not form functional homomeric channels, but participates in the formation of heteromeric complexes with other potassium α-subunits when expressed in Xenopus oocytes. The interaction of KDC1 with KAT1 was investigated using the yeast two-hybrid system, fluorescence and electrophysiological studies. We found that the KDC1-EGFP fusion protein is not targeted to the plasma membrane of Xenopus oocytes unless it is coexpressed with KAT1. Deletion mutants revealed that the KDC1 C-terminus is involved in heteromerization. Two domains of the C-terminus, the region downstream the putative cyclic nucleotide binding domain and the distal part of the C-terminus called K_HA domain, contributed to a different extent to channel assembly. Whereas the first interacting region of the C-terminus was necessary for channel heteromerization, the removal of the distal K_HA domain decreased but did not abolish the formation of heteromeric complexes. Similar results were obtained when coexpressing KDC1 with the KAT1-homolog KDC2 from carrots, thus indicating the physiological significance of the KAT1/KDC1 characterization. Electrophysiological experiments showed furthermore that the heteromerization capacity of KDC1 was negatively influenced by the presence of the enhanced green fluorescence protein fusion.     

Histidines Are Responsible for Zinc Potentiation of the Current in KDC1 Carrot Channels

Unlike all plant inward-rectifying potassium channels, the carrot channel KDC1 has two histidine pairs (H161,H162) in the S3–S4 and (H224,H225) in the S5–S6 linkers. When coinjected with KAT1 in Xenopus oocytes, KDC1 participates in the formation of heteromultimeric KDC1:KAT1 channels and the ionic current is potentiated by extracellular Zn²⁺. To investigate the potential interactions between KDC1 and zinc, a KDC1-KAT1 dimer was constructed. The dimeric and heteromeric channels displayed similar characteristics and the same sensitivity to zinc and other metals; this result suggests that zinc binding is mediated by residues in a single channel subunit. The KDC1:KAT1 currents were also potentiated by external Pb²⁺ and Cd²⁺ and inhibited by Ni²⁺. To investigate further the role of KDC1-histidines, these amino acids were mutated into alanines. The single mutations H225A, H161A, and H162A did not affect the response of the heteromeric channels to zinc. Conversely, the single mutant H224A and the double mutants (H224A,H225A) and (H161A,H162A) abolished zinc potentiation, but not that induced by Pb²⁺ or Cd²⁺. These results suggest that Zn²⁺ potentiation cannot be ascribed to simple electrostatic interactions between zinc and channel residues and that histidine 224 is crucial for zinc but not for lead potentiation of the current.     

Preferential KAT1-KAT2 Heteromerization Determines Inward K+ Current Properties in Arabidopsis Guard Cells

Guard cells adjust their volume by changing their ion content due to intense fluxes that, for K⁺, are believed to flow through inward or outward Shaker channels. Because Shaker channels can be homo- or heterotetramers and Arabidopsis guard cells express at least five genes encoding inward Shaker subunits, including the two major ones, KAT1 and KAT2, the molecular identity of inward Shaker channels operating therein is not yet completely elucidated. Here, we first addressed the properties of KAT1-KAT2 heteromers by expressing KAT1-KAT2 tandems in Xenopus oocytes. Then, computer analyses of the data suggested that coexpression of free KAT1 and KAT2 subunits resulted mainly in heteromeric channels made of two subunits of each type due to some preferential association of KAT1-KAT2 heterodimers at the first step of channel assembly. This was further supported by the analysis of KAT2 effect on KAT1 targeting in tobacco cells. Finally, patch-clamp recordings of native inward channels in wild-type and mutant genotypes strongly suggested that this preferential heteromerization occurs in planta and that Arabidopsis guard cell inward Shaker channels are mainly heteromers of KAT1 and KAT2 subunits.     

Increased functional diversity of plant K+ channels by preferential heteromerization of the shaker-like subunits AKT2 and KAT2

Assembly of plant Shaker subunits as heterotetramers, increasing channel functional diversity, has been reported. Here we focus on a new interaction, between AKT2 and KAT2 subunits. The assembly as AKT2/KAT2 heterotetramers is demonstrated by (i) a strong signal in two-hybrid tests with intracytoplasmic C-terminal regions, (ii) the effect of KAT2 on AKT2 subunit targeting in tobacco cells, (iii) the complete inhibition of AKT2 currents by co-expression with a dominant-negative KAT2 subunit in Xenopus oocytes, and reciprocally, and (iv) the appearance, upon co-expression of wild-type AKT2 and KAT2 subunits, of new channel functional properties that cannot be explained by the co-existence of two kinds of homotetrameric channels. In particular, the instantaneous current, characteristic of AKT2, displayed new functional features when compared with those of AKT2 homotetramers: activation by external acidification (instead of inhibition) and weak inhibition by calcium. Single channel current measurements in oocytes co-expressing AKT2 and KAT2 revealed a strong preference for incorporation of subunits into heteromultimers and a diversity of individual channels. In planta, these new channels, which may undergo specific regulations, are likely to be formed in guard cells and in the phloem, where they could participate in the control of membrane potential and potassium fluxes.     

A Novel K+ Channel Expressed in Carrot Roots with a Low Susceptibility Toward Metal Ions

Kdc1 is a novel K⁺-channel gene cloned from carrot roots, and which is also present in cultured carrot cells. We investigated the characteristics of the ionic current elicited in Xenopus oocytes coinjected with KDC1 (K⁺-Daucus carota 1) and KAT1 (from Arabidopsis thaliana) RNA. Expressed heteromeric channels displayed inward-rectifying potassium currents whose kinetics, voltage characteristics, and inhibition by metal ions depended on KDC1:KAT1 ratios. At low KDC1:KAT1 ratios, Zn²⁺ inhibition of heteromeric K⁺ current was less pronounced compared to homomeric KAT1 channels, while at higher KDC1:KAT1 ratios, the addition of Zn²⁺ even produced an increase in current. Under the same conditions, the Ni²⁺ inhibition of the current was also reduced, but no current increase was observed. These effects might be explained by the unusual amino acid composition of the KDC1 protein in terms of histidine residues that are absent in the pore region, but abundant (four per subunit) in the proximity of the pore entrance. Channels like KDC1 could be at least partially responsible for the higher resistance of carrot cells in the presence of metals.     

Molecular coupling between voltage sensor and pore opening in the Arabidopsis inward rectifier K+ channel KAT1

Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open.     

AtKC1, a conditionally targeted Shaker-type subunit, regulates the activity of plant K+ channels

Amongst the nine voltage-gated K⁺ channel (Kv) subunits expressed in Arabidopsis, AtKC1 does not seem to form functional Kv channels on its own, and is therefore said to be silent. It has been proposed to be a regulatory subunit, and to significantly influence the functional properties of heteromeric channels in which it participates, along with other Kv channel subunits. The mechanisms underlying these properties of AtKC1 remain unknown. Here, the transient (co-)expression of AtKC1 , AKT1 and/or KAT1 genes was obtained in tobacco mesophyll protoplasts, which lack endogenous inward Kv channel activity. Our experimental conditions allowed both localization of expressed polypeptides (GFP-tagging) and recording of heterologously expressed Kv channel activity (untagged polypeptides). It is shown that AtKC1 remains in the endoplasmic reticulum unless it is co-expressed with AKT1. In these conditions heteromeric AtKC1-AKT1 channels are obtained, and display functional properties different from those of homomeric AKT1 channels in the same context. In particular, the activation threshold voltage of the former channels is more negative than that of the latter ones. Also, it is proposed that AtKC1-AKT1 heterodimers are preferred to AKT1-AKT1 homodimers during the process of tetramer assembly. Similar results are obtained upon co-expression of AtKC1 with KAT1 . The whole set of data provides evidence that AtKC1 is a conditionally-targeted Kv subunit, which probably downregulates the physiological activity of other Kv channel subunits in Arabidopsis.     

Biochemical characterization of the Arabidopsis K+ channels KAT1 and AKT1 expressed or co-expressed in insect cells

KAT1 and AKT1 belong to the multigenic family of the inwardly rectifying Shaker-like plant K+ channels. They were biochemically characterized after expression in insect cells using recombinant baculoviruses. The channels were solubilized from microsomal fractions prepared from infected cells (among eight different detergents only one, L-alpha-lysophosphatidylcholine, was efficient for solubilization), and purified to homogeneity using immunoaffinity (KAT1) or ion-exchange and size exclusion (AKT1) techniques. The following results were obtained with the purified polypeptides: (i) neither KAT1 nor AKT1 was found to be glycosylated; (ii) both polypeptides were mainly present as homotetrameric structures, supporting the hypothesis of a tetrameric structure for the functional channels; (iii) no heteromeric KAT1/AKT1 assembly was detected when the two polypeptides were co-expressed in insect cells. The use of the two-hybrid system in yeast also failed to detect any interaction between KAT1 and AKT1 polypeptides. Because of these negative results, the hypothesis that plant K+-channel subunits are able to co-assemble without any discrimination, previously put forward based on co-expression in Xenopus oocytes of various K+-channel subunits (including KAT1 and AKT1), has still to be supported by independent approaches. Co-localization of channel subunits within the same plant tissue/cell does not allow us to conclude that the subunits form heteromultimeric channels.     

AtKC1, a conditionally targeted Shaker-type subunit, regulates the activity of plant K+ channels.

Amongst the nine voltage-gated K(+) channel (Kv) subunits expressed in Arabidopsis, AtKC1 does not seem to form functional Kv channels on its own, and is therefore said to be silent. It has been proposed to be a regulatory subunit, and to significantly influence the functional properties of heteromeric channels in which it participates, along with other Kv channel subunits. The mechanisms underlying these properties of AtKC1 remain unknown. Here, the transient (co-)expression of AtKC1, AKT1 and/or KAT1 genes was obtained in tobacco mesophyll protoplasts, which lack endogenous inward Kv channel activity. Our experimental conditions allowed both localization of expressed polypeptides (GFP-tagging) and recording of heterologously expressed Kv channel activity (untagged polypeptides). It is shown that AtKC1 remains in the endoplasmic reticulum unless it is co-expressed with AKT1. In these conditions heteromeric AtKC1-AKT1 channels are obtained, and display functional properties different from those of homomeric AKT1 channels in the same context. In particular, the activation threshold voltage of the former channels is more negative than that of the latter ones. Also, it is proposed that AtKC1-AKT1 heterodimers are preferred to AKT1-AKT1 homodimers during the process of tetramer assembly. Similar results are obtained upon co-expression of AtKC1 with KAT1. The whole set of data provides evidence that AtKC1 is a conditionally-targeted Kv subunit, which probably downregulates the physiological activity of other Kv channel subunits in Arabidopsis.     

KDC1, a carrot Shaker-like potassium channel, reveals its role as a silent regulatory subunit when expressed in plant cells

The Shaker potassium channels are tetrameric proteins formed by the assembly of four α-subunits. The oligomerization can occur among both homo- and hetero-α-subunits. KDC1 is a carrot Shaker-like potassium channel expressed in the epidermis of plantlet roots and the protoderm of somatic embryos. KDC1 was previously characterised electrophysiologically in CHO and Xenopus oocytes cells, but the experiments performed in these systems did not provide conclusive evidence that KDC1 forms a functional homomeric channel in plant cells. In this report, we show that KDC1 localizes to the plasma membrane of root cells in transgenic tobacco plants transformed with a KDC1∷GFP fusion construct. In tobacco mesophyll protoplasts, transiently transformed with KDC1∷GFP, KDC1 was present on the endomembrane and the protoplasts did not show any inward potassium current, as demonstrated by patch-clamp experiments. The co-expression of KDC1∷GFP with the Arabidopsis thaliana potassium channel AKT1 in tobacco mesophyll protoplasts has the effect of shifting KDC1 localization from endomembranes to the plasma membrane. Patch-clamp experiments performed on tobacco mesophyll protoplasts expressing AKT1 alone or in combination with KDC1∷GFP showed voltage-activated inward potassium currents with different properties. In particular, the addition of Zn²⁺ to the bath solution induced a clear decrease of the potassium currents in protoplasts transformed with AKT1 alone, whereas a current potentiation (indicative of KDC1 presence) was observed in protoplasts co-transformed with AKT1 + KDC1∷GFP. Split-Ubiquitin assay experiments performed in yeast cells confirmed the interaction between AKT1 and KDC1.     

Expanded Functional Diversity of Shaker K+ Channels in Cnidarians Is Driven by Gene Expansion

The genome of the cnidarian Nematostella vectensis (starlet sea anemone) provides a molecular genetic view into the first nervous systems, which appeared in a late common ancestor of cnidarians and bilaterians. Nematostella has a surprisingly large and diverse set of neuronal signaling genes including paralogs of most neuronal signaling molecules found in higher metazoans. Several ion channel gene families are highly expanded in the sea anemone, including three subfamilies of the Shaker K⁺ channel gene family: Shaker (Kv1), Shaw (Kv3) and Shal (Kv4). In order to better understand the physiological significance of these voltage-gated K⁺ channel expansions, we analyzed the function of 18 members of the 20 gene Shaker subfamily in Nematostella. Six of the Nematostella Shaker genes express functional homotetrameric K⁺ channels in vitro. These include functional orthologs of bilaterian Shakers and channels with an unusually high threshold for voltage activation. We identified 11 Nematostella Shaker genes with a distinct “silent” or “regulatory” phenotype; these encode subunits that function only in heteromeric channels and serve to further diversify Nematostella Shaker channel gating properties. Subunits with the regulatory phenotype have not previously been found in the Shaker subfamily, but have evolved independently in the Shab (Kv2) family in vertebrates and the Shal family in a cnidarian. Phylogenetic analysis indicates that regulatory subunits were present in ancestral cnidarians, but have continued to diversity at a high rate after the split between anthozoans and hydrozoans. Comparison of Shaker family gene complements from diverse metazoan species reveals frequent, large scale duplication has produced highly unique sets of Shaker channels in the major metazoan lineages.     

Subunit Stoichiometry of Human Muscle Chloride Channels

Voltage-gated Cl⁻ channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl⁻ channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl⁻ channels exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.     

Inward rectifier potassium channels in plants differ from their animal counterparts in response to voltage and channel modulators

We have investigated the electrophysiological basis of potassium inward rectification of the KAT1 gene product from Arabidopsis thaliana expressed in Xenopus oocytes and of functionally related K⁺ channels in the plasma membrane of guard and root cells from Vicia faba and Zea mays. The whole-cell currents passed by these channels activate, following steps to membrane potentials more negative than –100 mV, with half activation times of tens of milliseconds. This voltage dependence was unaffected by the removal of cytoplasmic magnesium. Consequently, unlike inward rectifier channels of animals, inward rectification of plant potassium channels is an intrinsic property of the channel protein itself. We also found that the activation kinetics of KAT1 were modulated by external pH. Decreasing the pH in the range 8.5 to 4.5 hastened activation and shifted the steady state activation curve by 19 mV per pH unit. This indicates that the activity of these K⁺ channels and the activity of the plasma membrane H⁺-ATPase may not only be coordinated by membrane potential but also by pH. The instantaneous current-voltage relationship, on the other hand, did not depend on pH, indicating that H⁺ do not block the channel. In addition to sensitivity towards protons, the channels showed a high affinity voltage dependent block in the presence of cesium, but were less sensitive to barium. Recordings from membrane patches of KAT1 injected oocytes in symmetric, Mg²⁺-free, 100 mM-K⁺, solutions allowed measurements of the current-voltage relation of single open KAT1 channels with a unitary conductance of 5 pS. We conclude that the inward rectification of the currents mediated by the KAT1 gene product, or the related endogenous channels of plant cells, results from voltage-modulated structural changes within the channel proteins. The voltage-sensing or the gating-structures appear to interact with a titratable acidic residue exposed to the extracellular medium. Correspondence to: R. Hedrich     

Evaluation of functional interaction between K(+) channel alpha- and beta-subunits and putative inactivation gating by Co-expression in Xenopus laevis oocytes

Animal K⁺ channel α- (pore-forming) subunits form native proteins by association with β-subunits, which are thought to affect channel function by modifying electrophysiological parameters of currents (often by inducing fast inactivation) or by stabilizing the protein complex. We evaluated the functional association of KAT1, a plant K⁺ channel α-subunit, and KAB1 (a putative homolog of animal K⁺ channel β-subunits) by co-expression in Xenopus laevis oocytes. Oocytes expressing KAT1 displayed inward-rectifying, non-inactivating K⁺ currents that were similar in magnitude to those reported in prior studies. K⁺ currents recorded from oocytes expressing both KAT1 and KAB1 had similar gating kinetics. However, co-expression resulted in greater total current, consistent with the possibility that KAB1 is a β-subunit that stabilizes and therefore enhances surface expression of K⁺ channel protein complexes formed by α-subunits such as KAT1. K⁺ channel protein complexes formed by α-subunits such as KAT1 that undergo (voltage-dependent) inactivation do so by means of a “ball and chain” mechanism; the ball portion of the protein complex (which can be formed by the N terminus of either an α- or β-subunit) occludes the channel pore. KAT1 was co-expressed in oocytes with an animal K⁺ channel α-subunit (hKv1.4) known to contain the N-terminal ball and chain. Inward currents through heteromeric hKv1.4:KAT1 channels did undergo typical voltage-dependent inactivation. These results suggest that inward currents through K⁺ channel proteins formed at least in part by KAT1 polypeptides are capable of inactivation, but the structural component facilitating inactivation is not present when channel complexes are formed by either KAT1 or KAB1 in the absence of additional subunits.     

Functional expression of the plant K channel KAT1 in insect cells

Following the biophysical analysis of plant K⁺ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1 and KST1 have recently been identified on the molecular level. Among them, we focused on the expression and characterization of the Arabidopsis thaliana K⁺ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K⁺-selective (impermeable to Na⁺ and even NH₄⁺) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as −60 to −80 mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca²⁺ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-translational modification and assembly of a green inward rectifier.     

Kvbeta1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to kv4.2 but not to Shaker channels.

Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic modulatory beta subunits. The differential expression and association of alpha and beta subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells, and their functional expression in heterologous systems provides a tool to study their regulation at a molecular level. Here, we have studied the effects of Kvbeta1.2 coexpression on the properties of Shaker and Kv4.2 KV channel alpha subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kvbeta1.2 functionally associates with these two alpha subunits, as well as with the endogenous KV channels of HEK293 cells, to modulate different properties of the heteromultimers. Kvbeta1.2 accelerates the rate of inactivation of the Shaker currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kvbeta1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2'-dithiodipyridine), which also modulate the low pO2 response of the Kv4.2+beta channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the effects of DTT. Finally, hypoxic inhibition of Kv4.2+beta currents can be reverted by 70% in the presence of carbon monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that beta subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability of Kv4. 2+beta channels to respond to hypoxia.     

Distinct Amino Acids in the C-Linker Domain of the Arabidopsis K+ Channel KAT2 Determine Its Subcellular Localization and Activity at the Plasma Membrane

Shaker K⁺ channels form the major K⁺ conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements, including a C-linker region and a cyclic nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the sixth transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis (Arabidopsis thaliana) Shaker subunits, K⁺ channel in Arabidopsis thaliana2 (KAT2) and Arabidopsis thaliana K⁺ rectifying channel1 (AtKC1). These two subunits contribute to K⁺ transport in planta by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane, whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified two contiguous amino acids, valine-381 and serine-382, located in the C-linker carboxy-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine-amino acid stretch ₃₁₂TVRAASEFA₃₂₀ that composes the first C-linker α-helix located just below the pore is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD three-dimensional model, based on animal HCN (for Hyperpolarization-activated, cyclic nucleotide-gated K⁺) channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function.In plants, potassium channels from the Shaker family dominate the plasma membrane (PM) conductance to K⁺ in most cell types and play crucial roles in sustained K⁺ transport (Blatt et al., 2012; Hedrich, 2012; Sharma et al., 2013). Plant Shaker channels, like their homologs in animals (Craven and Zagotta, 2006; Wahl-Schott and Biel, 2009), belong to the six transmembrane-one pore (6TM-1P) cation channel superfamily. Functional channels are tetrameric proteins arranged around a central pore (Daram et al., 1997; Urbach et al., 2000; Dreyer et al., 2004). These channels can result from the assembly of Shaker subunits encoded by the same gene (homotetramers) or by different Shaker genes (heterotetramers). Heterotetramerization has been extensively reported within the inwardly rectifying Shaker channel group (five members in Arabidopsis [Arabidopsis thaliana]) and increased channel functional diversity (Jeanguenin et al., 2008; Lebaudy et al., 2008a).Based on in silico sequence analyses, plant Shaker subunits display a short cytosolic N-terminal domain, followed by the 6TM-1P hydrophobic core, and a long C-terminal cytosolic region in which several domains can be identified. The first one, named C-linker (about 80 amino acids in length), is followed by a cyclic nucleotide-binding domain (CNBD), an ankyrin domain (involved in protein-protein interaction; Lee et al., 2007, Grefen and Blatt, 2012), and a domain named K_HA (Ehrhardt et al., 1997) rich in hydrophobic and acidic residues. Sequence analysis of plant Shaker channels indicates that, among these cytosolic domains, the highest levels of similarity are displayed by the C-linker and the CNBD domains. Interestingly, both domains are also highly conserved in some members from the animal K⁺ channel superfamily, like Hyperpolarization-activated, cyclic nucleotide-gated K⁺ channel (HCN), K⁺ voltage-gated channel, subfamily H (KCNH), and Cyclic-nucleotide-gated ion channel (CNGC). In these animal 6TM-1P channels, the roles of C-linker and CNBD domains have been extensively investigated via crystal structure analyses (Zagotta et al., 2003; Brelidze et al., 2012), whereas plant Shaker channels are still poorly characterized at the structural level (Dreyer et al., 2004; Gajdanowicz et al., 2009; Naso et al., 2009; Garcia-Mata et al., 2010).Aiming at investigating the structure-function relationship of plant Shaker channels, we have used the Arabidopsis Shaker subunit K⁺ channel in Arabidopsis thaliana2 (KAT2) as a model. We developed a subdomain-swapping strategy between KAT2 and another Shaker subunit displaying distinctive features, Arabidopsis thaliana K⁺ rectifying channel1 (AtKC1). The KAT2 subunit can form homomeric or heteromeric inwardly rectifying K⁺ channels at the PM and has been shown to be strongly expressed in guard cells, where it provides a major contribution to the membrane conductance to K⁺ (Pilot et al., 2001; Lebaudy et al., 2008b). In contrast, the behavior of AtKC1 is more complex. In planta, this subunit is coexpressed with other inwardly rectifying Shaker subunits, including KAT2, in different plant tissues (Jeanguenin et al., 2011), and in roots, direct evidence has been obtained that AtKC1 is involved in functional heterotetrameric channel formation with AKT1 (Reintanz et al., 2002; Honsbein et al., 2009). However, experiments performed in tobacco (Nicotiana tabacum) mesophyll protoplasts have revealed that when expressed alone, AtKC1 is entrapped in the endoplasmic reticulum (ER). However, in tobacco protoplasts and Xenopus laevis oocytes, coexpression of AtKC1 with KAT2 or other inwardly rectifying Shaker subunits (AKT1, KAT1, or AKT2) gives rise to functional heteromeric channels (Duby et al., 2008; Jeanguenin et al., 2011). In Arabidopsis, it is interesting that evidence of the AtKC1 retention in the ER compartment, in the absence of other Shaker subunits, is lacking, since in the native tissues, AtKC1 is always expressed with its inward partners, with which it is able to form heteromeric channels.Here, we took advantage of the unique behavior of AtKC1 when expressed in heterologous systems to investigate the structure-function relationship of the C-linker of KAT2 by sequence exchange between these two channel subunits and by site-directed mutagenesis. The C-linker domain, which, to our knowledge, had never been studied as such in plant Shaker channels before, could be predicted to play crucial roles in channel properties due to its strategic location between the channel transmembrane core and the cytoplasmic CNBD domain. The resulting KAT2-AtKC1 chimeras were expressed in tobacco mesophyll protoplasts and in X. laevis oocytes for investigating their subcellular localization and measuring their activity at the cell membrane. Here, we show that two amino acids present in the C-linker are important for channel subcellular location and that a stretch of nine amino acids forming a short helix just below the membrane, downstream of the sixth transmembrane segment of the channel hydrophobic core, is involved in channel gating. The obtained experimental results are discussed in relation with a KAT2 C-linker/CNBD three-dimensional (3D) model based on animal HCN channels as structure templates.     

Random assembly of SUR subunits in K(ATP) channel complexes

Sulfonylurea receptors (SURs) associate with Kir6.x subunits to form tetradimeric K(ATP) channel complexes. SUR1 and SUR2 confer differential channel sensitivities to nucleotides and pharmacological agents, and are expressed in specific, but overlapping, tissues. This raises the question of whether these different SUR subtypes can assemble in the same channel complex and generate channels with hybrid properties. To test this, we engineered dimeric constructs of wild type or N160D mutant Kir6.2 fused to SUR1 or SUR2A. Dimeric fusions formed functional, ATP-sensitive, channels. Coexpression of weakly rectifying SUR1-Kir6.2 (WTF-1) with strongly rectifying SUR1-Kir6.2[N160D] (NDF-1) in COSm6 cells results in mixed subunit complexes that exhibit unique rectification properties. Coexpression of NDF-1 and SUR2A-Kir6.2 (WTF-2) results in similar complex rectification, reflecting the presence of SUR1- and SUR2A-containing dimers in the same channel. The data demonstrate clearly that SUR1 and SUR2A subunits associate randomly, and suggest that heteromeric channels will occur in native tissues.