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.     

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.     

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.     

Stoichiometry studies reveal functional properties of KDC1 in plant shaker potassium channels

Functional heteromeric plant Shaker potassium channels can be formed by the assembly of subunits from different tissues, as well as from diverse plant species. KDC1 (K(+) Daucus carota 1) produces inward-rectifying currents in Xenopus oocytes when coexpressed with KAT1 and other subunits appertaining to different plant Shaker subfamilies. Owing to the presence of KDC1, resulting heteromeric channels display slower activation kinetics, a shift of the activation threshold toward more negative membrane potentials and current potentiation upon the addition of external zinc. Despite available information on heteromerization of plant Shaker channels, very little is known to date on the properties of the various stoichiometric configurations formed by different subunits. To investigate the functional properties of heteromeric nKDC1/mKAT1 configurations, we realized a series of dimeric constructs combining KDC1 and KAT1 alpha-subunits. We found that homomeric channels, formed by monomeric or dimeric alpha-subunit constructs, show identical biophysical characteristics. Coinjections of diverse tandem constructs, instead, displayed significantly different currents proving that KDC1 has high affinity for KAT1 and participates in the formation of functional channels with at most two KDC1 subunits, whereas three KDC1 subunits prevented the formation of functional channels. This article brings a contribution to the understanding of the molecular mechanisms regulating plant Shaker channel functionality by association of modulatory 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.     

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.     

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.     

Plant K+ channel alpha-subunits assemble indiscriminately.

In plants a large diversity of inwardly rectifying K+ channels (K(in) channels) has been observed between tissues and species. However, only three different types of voltage-dependent plant K+ uptake channel subfamilies have been cloned so far; they relate either to KAT1, AKT1, or AtKC1. To explore the mechanisms underlying the channel diversity, we investigated the assembly of plant inwardly rectifying alpha-subunits. cRNA encoding five different K+ channel alpha-subunits of the three subfamilies (KAT1, KST1, AKT1, SKT1, and AtKC1) which were isolated from different tissues, species, and plant families (Arabidopsis thaliana and Solanum tuberosum) was reciprocally co-injected into Xenopus oocytes. We identified plant K+ channels as multimers. Moreover, using K+ channel mutants expressing different sensitivities to voltage, Cs+, Ca2+, and H+, we could prove heteromers on the basis of their altered voltage and modulator susceptibility. We discovered that, in contrast to animal K+ channel alpha-subunits, functional aggregates of plant K(in) channel alpha-subunits assembled indiscriminately. Interestingly, AKT-type channels from A. thaliana and S. tuberosum, which as homomers were electrically silent in oocytes after co-expression, mediated K+ currents. Our findings suggest that K+ channel diversity in plants results from nonselective heteromerization of different alpha-subunits, and thus depends on the spatial segregation of individual alpha-subunit pools and the degree of temporal overlap and kinetics of expression.     

The potassium channel KAT1 is activated by plant and animal 14-3-3 proteins

14-3-3 proteins modulate the plant inward rectifier K+ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by +11 mV and increased the tau(on). KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (approximately 70% at -150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.     

Multiple genes, tissue specificity, and expression-dependent modulationcontribute to the functional diversity of potassium channels in Arabidopsis thaliana.

K+ channels play diverse roles in mediating K+ transport and in modulating the membrane potential in higher plant cells during growth and development. Some of the diversity in K+ channel functions may arise from the regulated expression of multiple genes encoding different K+ channel polypeptides. Here we report the isolation of a novel Arabidopsis thaliana cDNA (AKT2) that is highly homologous to the two previously identified K+ channel genes, KAT1 and AKT1. This cDNA mapped to the center of chromosome 4 by restriction fragment length polymorphism analysis and was highly expressed in leaves, whereas AKT1 was mainly expressed in roots. In addition, we show that diversity in K+ channel function may be attributable to differences in expression levels. Increasing KAT1 expression in Xenopus oocytes by polyadenylation of the KAT1 mRNA increased the current amplitude and led to higher levels of KAT1 protein, as assayed in western blots. The increase in KAT1 expression in oocytes produced shifts in the threshold potential for activation to more positive membrane potentials and decreased half-activation times. These results suggest that different levels of expression and tissue-specific expression of different K+ channel isoforms can contribute to the functional diversity of plant K+ channels. The identification of a highly expressed, leaf-specific K+ channel homolog in plants should allow further molecular characterization of K+ channel functions for physiological K+ transport processes in leaves.     

The N-terminus of the K channel KAT1 controls its voltage-dependent gating by altering the membrane electric field.

Functional roles of different domains (pore region, S4 segment, N-terminus) of the KAT1 potassium channel in its voltage-dependent gating were electrophysiologically studied in Xenopus oocytes. The KAT1 properties did not depend on the extracellular K+ concentration or on residue H267, equivalent to one of the residues known to be important in C-type inactivation in Shaker channels, indicating that the hyperpolarization-induced KAT1 inward currents are related to the channel activation rather than to recovery from inactivation. Neutralization of a positively charged amino acid in the S4 domain (R176S) reduced the gating charge movement, suggesting that it acts as a voltage-sensing residue in KAT1. N-terminal deletions alone (e.g., delta20-34) did not affect the gating charge movement. However, the deletions paradoxically increased the voltage sensitivity of the R176S mutant channel, but not that of the wild-type channel. We propose a simple model in which the N-terminus determines the KAT1 voltage sensitivity by contributing to the electric field sensed by the voltage sensor.     

The K+ channel KZM2 is involved in stomatal movement by modulating inward K+ currents in maize guard cells

Stomata are the major gates in plant leaf that allow water and gas exchange, which is essential for plant transpiration and photosynthesis. Stomatal movement is mainly controlled by the ion channels and transporters in guard cells. In Arabidopsis, the inward Shaker K⁺ channels, such as KAT1 and KAT2, are responsible for stomatal opening. However, the characterization of inward K⁺ channels in maize guard cells is limited. In the present study, we identified two KAT1‐like Shaker K⁺ channels, KZM2 and KZM3, which were highly expressed in maize guard cells. Subcellular analysis indicated that KZM2 and KZM3 can localize at the plasma membrane. Electrophysiological characterization in HEK293 cells revealed that both KZM2 and KZM3 were inward K⁺ (K_in) channels, but showing distinct channel kinetics. When expressed in Xenopus oocytes, only KZM3, but not KZM2, can mediate inward K⁺ currents. However, KZM2 can interact with KZM3 forming heteromeric K_in channel. In oocytes, KZM2 inhibited KZM3 channel conductance and negatively shifted the voltage dependence of KZM3. The activation of KZM2–KZM3 heteromeric channel became slower than the KZM3 channel. Patch‐clamping results showed that the inward K⁺ currents of maize guard cells were significantly increased in the KZM2 RNAi lines. In addition, the RNAi lines exhibited faster stomatal opening after light exposure. In conclusion, the presented results demonstrate that KZM2 functions as a negative regulator to modulate the K_in channels in maize guard cells. KZM2 and KZM3 may form heteromeric K_in channel and control stomatal opening in maize.     

Probing the pharmacological properties of distinct subunit interfaces within heteromeric glycine receptors reveals a functional ββ agonist-binding site

Synaptic glycine receptors (GlyRs) are hetero-pentameric chloride channels composed of α and β subunits, which are activated by agonist binding at subunit interfaces. To examine the pharmacological properties of each potential agonist-binding site, we substituted residues of the GlyR α(1) subunit by the corresponding residues of the β subunit, as deduced from sequence alignment and homology modeling based on the recently published crystal structure of the glutamate-gated chloride channel GluCl. These exchange substitutions allowed us to reproduce the βα, αβ and ββ subunit interfaces present in synaptic heteromeric GlyRs by generating recombinant homomeric receptors. When the engineered α(1) GlyR mutants were expressed in Xenopus oocytes, all subunit interface combinations were found to form functional agonist-binding sites as revealed by voltage clamp recording. The ββ-binding site displayed the most distinct pharmacological profile towards a range of agonists and modulators tested, indicating that it might be selectively targeted to modulate the activity of synaptic GlyRs. The mutational approach described here should be generally applicable to heteromeric ligand-gated ion channels composed of homologous subunits and facilitate screening efforts aimed at targeting inter-subunit specific binding sites.     

Guard cell inward K+ channel activity in arabidopsis involves expression of the twin channel subunits KAT1 and KAT2

Stomatal opening, which controls gas exchanges between plants and the atmosphere, results from an increase in turgor of the two guard cells that surround the pore of the stoma. KAT1 was the only inward K(+) channel shown to be expressed in Arabidopsis guard cells, where it was proposed to mediate a K(+) influx that enables stomatal opening. We report that another Arabidopsis K(+) channel, KAT2, is expressed in guard cells. More than KAT1, KAT2 displays functional features resembling those of native inward K(+) channels in guard cells. Coexpression in Xenopus oocytes and two-hybrid experiments indicated that KAT1 and KAT2 can form heteromultimeric channels. The data indicate that KAT2 plays a crucial role in the stomatal opening machinery.     

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.     

Regulation by external K+ in a maize inward shaker channel targets transport activity in the high concentration range

An inward Shaker K(+) channel identified in Zea mays (maize), ZmK2.1, displays strong regulation by external K(+) when expressed in Xenopus laevis (African clawed frog) oocytes or COS cells. ZmK2.1 is specifically activated by K(+) with an apparent K(m) close to 15 mM independent of the membrane hyperpolarization level. In the absence of K(+), ZmK2.1 appears to enter a nonconducting state. Thus, whatever the membrane potential, this maize channel cannot mediate K(+) influx in the submillimolar concentration range, unlike its relatives in Arabidopsis thaliana. Its expression is restricted to the shoots, the strongest signal (RT-PCR) being associated with vascular/bundle sheath strands. Based on sequence and gene structure, the closest relatives of ZmK2.1 in Arabidopsis are K(+) Arabidopsis Transporter 1 (KAT1) (expressed in guard cells) and KAT2 (expressed in guard cells and leaf phloem). Patch-clamp analyses of guard cell protoplasts reveal a higher functional diversity of K(+) channels in maize than in Arabidopsis. Channels endowed with regulation by external K(+) similar to that of ZmK2.1 (channel activity regulated by external K(+) with a K(m) close to 15 mM, regulation independent of external Ca(2+)) constitute a major component of the maize guard cell inward K(+) channel population. The presence of such channels in maize might reflect physiological traits of C4 and/or monocotyledonous plants.     

Gating of heteromeric retinal rod channels by cyclic AMP: role of the C-terminal and pore domains

Cyclic nucleotide-gated channels are tetramers composed of homologous alpha and beta subunits. C-terminal truncation mutants of the alpha and beta subunits of the retinal rod channel were expressed in Xenopus oocytes, and analyzed for cGMP- and cAMP-induced currents (single-channel records and macroscopic currents). When the alpha subunit truncated downstream of the cGMP-binding site (alpha D608stop) is co-injected with truncated beta subunits, the heteromeric channels present a drastic increase of cAMP sensitivity. A partial effect is observed with heteromeric alpha R656stop-containing channels, while alpha K665stop-containing channels behave like alpha wt/beta wt. The three truncated alpha subunits have wild-type activity when expressed alone. Heteromeric channels composed of alpha wt or truncated alpha subunits and chimeric beta subunits containing the pore domain of the alpha subunit have the same cAMP sensitivity as alpha-only channels. The results disclose the key role of two domains distinct from the nucleotide binding site in the gating of heteromeric channels by cAMP: the pore of the beta subunit, which has an activating effect, and a conserved domain situated downstream of the cGMP-binding site in the alpha subunit (I609-K665), which inhibits this effect.     

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.