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.     

Potassium transport in developing fleshy fruits: the grapevine inward K+ channel VvK1.2 is activated by CIPK–CBL complexes and induced in ripening berry flesh cells

The grape berry provides a model for investigating the physiology of non‐climacteric fruits. Increased K⁺ accumulation in the berry has a strong negative impact on fruit acidity (and quality). In maturing berries, we identified a K⁺ channel from the Shaker family, VvK1.2, and two CBL‐interacting protein kinase (CIPK)/calcineurin B‐like calcium sensor (CBL) pairs, VvCIPK04–VvCBL01 and VvCIPK03–VvCBL02, that may control the activity of this channel. VvCBL01 and VvCIPK04 are homologues of Arabidopsis AtCBL1 and AtCIPK23, respectively, which form a complex that controls the activity of the Shaker K⁺ channel AKT1 in Arabidopsis roots. VvK1.2 remained electrically silent when expressed alone in Xenopus oocytes, but gave rise to K⁺ currents when co‐expressed with the pairs VvCIPK03–VvCBL02 or VvCIPK04–VvCBL01, the second pair inducing much larger currents than the first one. Other tested CIPK–CBL pairs expressed in maturing berries were found to be unable to activate VvK1.2. When activated by its CIPK–CBL partners, VvK1.2 acts as a voltage‐gated inwardly rectifying K⁺ channel that is activated at voltages more negative than –100 mV and is stimulated upon external acidification. This channel is specifically expressed in the berry, where it displays a very strong induction at veraison (the inception of ripening) in flesh cells, phloem tissues and perivascular cells surrounding vascular bundles. Its expression in these tissues is further greatly increased upon mild drought stress. VvK1.2 is thus likely to mediate rapid K⁺ transport in the berry and to contribute to the extensive re‐organization of the translocation pathways and transport mechanisms that occurs at veraison.     

External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism

Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell. This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants.     

Intracellular K+ sensing of SKOR, a Shaker-type K+ channel from Arabidopsis

Most K+ channels in plants are structurally classified into the Shaker family named after the shaker K+ channel in Drosophila. Plant K+ channels function in many physiological processes including osmotic regulation and K+ nutrition. An outwardly rectifying K+ channel, SKOR, mediates the delivery of K+ from stelar cells to the xylem in the roots, a critical step in the long-distance distribution of K+ from roots to the upper parts of the plant. Here we report that SKOR channel activity is strictly dependent on intracellular K+ concentrations. Activation by K+ did not affect the kinetics of voltage dependence in SKOR, indicating that a voltage-independent gating mechanism underlies the K+ sensing process. Further analysis showed that the C-terminal non-transmembrane region of the SKOR protein was required for this sensing process. The intracellular K+ sensing mechanism couples SKOR activity to K+ nutrition status in the 'source cells', thereby establishing a supply-based unloading system for the regulation of K+ distribution.     

The K+ channel SKT1 is co-expressed with KST1 in potato guard cells--both channels can co-assemble via their conserved KT domains

An appreciable number of potassium channels mediating K+ uptake have been identified in higher plants. Promoter-beta-glucuronidase reporter gene studies were used here to demonstrate that SKT1, encoding a potato K+ inwardly rectifying channel, is expressed in guard cells in addition to KST1 previously reported. However, whereas KST1 was found to be expressed in essentially all mature guard cells, SKT1 expression was almost exclusively restricted to guard cells of the abaxial leaf epidermis. This suggests that different types of K+ channel subunits contribute to channel formation in potato guard cells and therefore differential regulation of stomatal movements in the two leaf surfaces. The overlapping expression pattern of SKT1 and KST1 in abaxial guard cells indicates that K+in channels of different sub-families contribute to ionic currents in this cell type, thus explaining the different properties of channels expressed solely in heterologous systems and those endogenous to guard cells. Interaction studies had previously suggested that plant K+ inward rectifiers form clusters via their conserved C-terminal domain, KT/HA. K+ channels co-expressed in one cell type may therefore form heteromers, which increase functional variability of K+ currents, a phenomenon well described for animal voltage-gated K+ channels. Co-expression of KST1 and SKT1 in Xenopus oocytes resulted in currents with an intermediate sensitivity towards Cs+, suggesting the presence of heteromers, and a sensitivity towards external Ca2+, which reflected the property of the endogenous K+in current in guard cells. Modulation of KST1 currents in oocytes by co-expressing KST1 with a SKT1 pore-mutant, which by itself was not able to confer activating K+ currents, demonstrated the possibility that KST1 and SKT1 co-assemble to hetero-oligomers. Furthermore, various C-terminal deletions of the mutated SKT1 channel restored KST1 currents, showing that the C-terminal KT motif is essential for heteromeric channel formation.     

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.     

Phosphatidic acid directly binds with rice potassium channel OsAKT2 to inhibit its activity

The plant Shaker K⁺ channel AtAKT2 has been identified as a weakly rectifying channel that can stabilize membrane potentials to promote photoassimilate phloem loading and translocation. Thus, studies on functional characterization and regulatory mechanisms of AtAKT2‐like channels in crops are highly important for improving crop production. Here, we identified the rice OsAKT2 as the ortholog of Arabidopsis AtAKT2, which is primarily expressed in the shoot phloem and localized at the plasma membrane. Using an electrophysiological assay, we found that OsAKT2 operated as a weakly rectifying K⁺ channel, preventing H⁺/sucrose‐symport‐induced membrane depolarization. Three critical amino acid residues (K193, N206, and S326) are essential to the phosphorylation‐mediated gating change of OsAKT2, consistent with the roles of the corresponding sites in AtAKT2. Disruption of OsAKT2 results in delayed growth of rice seedlings under short‐day conditions. Interestingly, the lipid second messenger phosphatidic acid (PA) inhibits OsAKT2‐mediated currents (both instantaneous and time‐dependent components). Lipid dot‐blot assay and liposome‐protein binding analysis revealed that PA directly bound with two adjacent arginine residues in the ANK domain of OsAKT2, which is essential to PA‐mediated inhibition of OsAKT2. Electrophysiological and phenotypic analyses also showed the PA‐mediated inhibition of AtAKT2 and the negative correlation between intrinsic PA level and Arabidopsis growth, suggesting that PA may inhibit AKT2 function to affect plant growth and development. Our results functionally characterize the Shaker K⁺ channel OsAKT2 and reveal a direct link between phospholipid signaling and plant K⁺ channel modulation.     

Molecular movement of the voltage sensor in a K channel

The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 A) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 A) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model.     

ER Export of KAT1 is Correlated to the Number of Acidic Residues within a Triacidic Motif

For a number of ion channels, including the potassium (K⁺) inward rectifying channel from Arabidopsis thaliana (KAT1), diacidic endoplasmic reticulum (ER) export motifs have been identified. These motifs consist of two acidic amino acids (aspartate (D) and/or glutamate (E)) separated by any amino acid. To specify the role of single acidic amino acids for efficiency of ER export, we analysed a sequence of KAT1 that included the originally identified diacidic ER export motif (DxE) plus an additional D just upstream of the diacidic motif. Analysis of single, double and triple mutations of the acidic amino acids of the DxDxE motif revealed a gradual reduction of ER export depending on the number of mutated acidic residues. The amount of reduction in ER export was not related to the position, but only to the number of mutated acidic amino acids. These results show that a triacidic motif is essential for efficient ER export of KAT1. Function of the triacidic motif probably involves cooperative binding to Sec24.     

Intracellular localization and physiological function of a rice Ca2+-permeable channel OsTPC1

Two-pore channels (TPCs) are cation channels with a voltage-sensor domain conserved in plants and animals. Rice OsTPC1 is predominantly localized to the plasma membrane (PM), and assumed to play an important role as a Ca²⁺-permeable cation channel in the regulation of cytosolic Ca²⁺ rise and innate immune responses including hypersensitive cell death and phytoalexin biosynthesis in cultured rice cells triggered by a fungal elicitor, xylanase from Trichoderma viride. In contrast, Arabidopsis AtTPC1 is localized to the vacuolar membrane (VM). To gain further insights into the intracellular localization of OsTPC1, we stably expressed OsTPC1-GFP in tobacco BY-2 cells. Confocal imaging and membrane fractionation revealed that, unlike in rice cells, the majority of OsTPC1-GFP fusion protein was targeted to the VM in tobacco BY-2 cells. Intracellular localization and functions of the plant TPC family is discussed.     

Two Novel Toxins from the Venom of the Scorpion Pandinus imperator Show that the N-terminal Amino Acid Sequence is Important for their Affinities towards Shaker B K+ Channels

Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K⁺ channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly block the Shaker B K⁺ channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas peptide Pi2, containing the Pro7, binds the Shaker B K⁺ channels with a K _d of 8.2 nm, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nm. Both peptides are capable of displacing the binding of ¹²⁵I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels. Received: 13 November 1995/Revised: 11 March 1996     

The short N-terminus is required for functional expression of the virus-encoded miniature K(+) channel Kcv

Kcv (K⁺ Chlorella virus) is a miniature virus-encoded K⁺ channel. Its predicted membrane–pore–membrane structure lacks a cytoplasmic C-terminus and it has a short 12 amino acid (aa) cytoplasmic N-terminus. Kcv forms a functional channel when expressed in human HEK 293 cells. Deletion of the 14 N-terminal aa results in no apparent differences in the subcellular location and expression level of the Kcv protein. However, the truncated protein does not induce a measurable current in transfected HEK 293 cells or Xenopus oocytes. We conclude that the N-terminus controls functional properties of the Kcv channel, but does not influence protein expression.     

Molecular cloning and characterization of LKv1, a novel voltage‐gated potassium channel in leech

We have cloned a novel voltage‐gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage‐gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I_K. Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage‐gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker‐related K channels in jellyfish. Thus, this analysis indicates that this group of voltage‐gated K channels contains several evolutionarily divergent lineages. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 287–299, 1999