Clustering of the K+ channel GORK of Arabidopsis parallels its gating by extracellular K+

GORK is the only outward‐rectifying Kv‐like K⁺ channel expressed in guard cells. Its activity is tightly regulated to facilitate K⁺ efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward‐rectifying K⁺ channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently‐tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K⁺ concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K⁺ channel blocker Ba²⁺. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo‐ and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K⁺, a characteristic that ensures the channel facilitates only K⁺ efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K⁺ above 1 mm . We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K⁺.     

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     

Evidence for a Multi-ion Pore Behavior in the Plant Potassium Channel KAT1

KAT1 is a cloned voltage-gated K⁺ channel from the plant Arabidopsis thaliana L., which displays an inward rectification reminiscent of `anomalous' rectification of the i <sub>f</sub> pacemaker current recorded in animal cells. Macroscopic conductance of KAT1 expressed in Xenopus oocytes was 5-fold less in pure Rb<sup>+</sup> solution than in pure K<sup>+</sup> solution, and negligible in pure Na<sup>+</sup> solution. Experiments in different K<sup>+</sup>/Na<sup>+</sup> or K<sup>+</sup>/Rb<sup>+</sup> mixtures revealed deviations from the principle of independence and notably two anomalous effects of the K<sup>+</sup>/Rb<sup>+</sup> mole fraction (i.e., the ratio [K<sup>+</sup>]/([K<sup>+</sup>]+[Rb<sup>+</sup>])). First, the KAT1 deactivation time constant was both voltage- and mole fraction-dependent (a so-called `foot in the door' effect was thus observed in KAT1 channel). Second, when plotted against the K⁺/Rb⁺ mole fraction, KAT1 conductance values passed through a minimum. This minimum is more important for two pore mutants of KAT1 (T259S and T260S) that displayed an increase in P_Rb/P_K. These results are consistent with the idea that KAT1 conduction requires several ions to be present simultaneously within the pore. Therefore, this atypical `green' member of the Shaker superfamily of K⁺ channels further shows itself to be an interesting model as well for permeation as for gating mechanism studies. Received: 9 February 1998/Revised: 28 July 1998     

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.     

Orientation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> KAT1 Channel in the Plasma Membrane

The Arabidopsis thaliana KAT1, an inward-rectifying potassium channel, shares molecular features with the Shaker family of outward rectifier K⁺ channels. The KAT1 amino-acid sequence reveals the presence of a positively charged S4 and a segment containing the TXGYGD signature sequence in the pore (P) region. To test whether the inward-rectifying properties of KAT1 are due to reverse orientation in the membrane, such that the voltage sensor is oriented in the opposite direction of the electric field compared with the Shaker K⁺ channel, we have inserted a flag epitope in the NH₂ terminus or the S3–S4 loop. The KAT1 and tagged constructs expressed functional channels in whole cells, Xenopus oocytes and COS-7. The electrophysiological properties of both tagged constructs were similar to those of the wild type. Immunofluorescence with an antibody against the flag epitope and an anti-C terminal KAT1 determined the membrane localization of these epitopes and the orientation of the KAT1 channel in the membrane. Our data confirm that KAT1 in eukaryotic cells has an orientation similar to the Shaker K⁺ channel.     

Increased Resistance to Extracellular Cation Block by Mutation of the Pore Domain of the Arabidopsis Inward-rectifying K+ Channel KAT1

Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K⁺ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K⁺ channel blockers are potent tools for studying the physiological functions and structural properties of K⁺ channels. In the present study the structural and biophysical mechanisms of Cs⁺ and TEA⁺ block of a cloned Arabidopsis inward-rectifying K⁺ channel (KAT1) were analyzed. Effects of the channel blockers Cs⁺ and TEA⁺ were characterized both extracellularly and intracellularly. Both external Cs⁺ and TEA⁺ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs⁺ and TEA⁺ than the wild-type K⁺ channel. Internal TEA⁺ and Cs⁺ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K⁺ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K⁺ channels. Received: 31 July 1995/Revised: 5 January 1996     

The Human Red Cell Voltage-regulated Cation Channel. The Interplay with the Chloride Conductance,the Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> Channel and the Ca<Superscript>2+</Superscript> Pump

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K⁺ channels. We report here the cloning of a K⁺ channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K⁺ channel characteristics. The amino-acid sequence of the eel K⁺ channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism.     

Complex interactions among residues within pore region determine the K+ dependence of a KAT1‐type potassium channel AmKAT1

KAT1‐type channels mediate K⁺ influx into guard cells that enables stomatal opening. In this study, a KAT1‐type channel AmKAT1 was cloned from the xerophyte Ammopiptanthus mongolicus. In contrast to most KAT1‐type channels, its activation is strongly dependent on external K⁺ concentration, so it can be used as a model to explore the mechanism for the K⁺‐dependent gating of KAT1‐type channels. Domain swapping between AmKAT1 and KAT1 reveals that the S5–pore–S6 region controls the K⁺ dependence of AmKAT1, and residue substitutions show that multiple residues within the S5–Pore linker and Pore are involved in its K⁺‐dependent gating. Importantly, complex interactions occur among these residues, and it is these interactions that determine its K⁺ dependence. Finally, we analyzed the potential mechanism for the K⁺ dependence of AmKAT1, which could originate from the requirement of K⁺ occupancy in the selectivity filter to maintain its conductive conformation. These results provide new insights into the molecular basis of the K⁺‐dependent gating of KAT1‐type channels.     

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.     

Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

Suppression of Inward-Rectifying K+ Channels KAT1 and AKT2 by Dominant Negative Point Mutations in the KAT1 α-Subunit

The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K⁺ (K⁺ _in ) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K⁺ _in channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K⁺ currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not greatly affect the ion selectivity of residual currents for Rb⁺, Na⁺, Li⁺, or Cs⁺. When T256R or G262K were co-expressed with a different K⁺ channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs⁺ block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers. These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued in vivo by the expression of the dominant negative K ⁺ _in channel mutants described here. Received: 2 July 1998/Revised: 23 October 1998     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Properties of the K+ inward rectifier in the plasma membrane of xylem parenchyma cells from barley roots: Effects of TEA+, Ca2+, Ba2+ and La3+

Xylem parenchyma cells are situated around the (apoplastic) xylem vessels and are involved in the control of the composition of the xylem sap by exporting and resorbing solutes. We investigated properties of the K⁺ inward rectifier in the plasma membrane of these cells by performing patch clamp experiments on protoplasts in the whole-cell configuration. Inward currents were sensitive to the K⁺ channel blocker TEA⁺ at a high concentration (20 mm). Barium, another classical K⁺ channel blocker, inhibited K⁺ currents with a K _i of about 1.3 mm. In contrast to guard cells, the cytosolic Ca²⁺ level proved to be ineffective in regulating the K⁺ conductance at hyperpolarization. External Ca²⁺ blocked currents weakly in a voltage-dependent manner. From instantaneous current-voltage curves, we identified a binding site in the channel pore with an electrical distance of about 0.2 to 0.5. Lanthanum ions reduced the inward current in a voltage-dependent manner and simultaneously displaced the voltage at which half of the channels are in the open state to more positive values. This finding was interpreted as resulting from a sum of two molecular effects, an interaction with the mouth of the channel that causes a reduction of current, and a binding to the voltage sensor, leading to a shielding of surface charges and, subsequently, a modulation of channel gating.A comparison between the K⁺ inward rectifier in xylem parenchyma cells, guard cells and KAT1 from Arabidopsis leads to the conclusion that these rectifiers form subtypes within one class of ion channels. The ineffectiveness of Ca²⁺ to control K⁺ influx in xylem parenchyma cells is interpreted in physiological terms.     

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.     

Inward and outward K+-selective currents in the plasma membrane of protoplasts from maize root cortex and stele

In an attempt to understand the processes mediating ion transport within the root, the patch clamp technique was applied to protoplasts isolated from the cortex and stele of maize roots and their plasma membrane conductances investigated. In the whole-cell configuration, membrane hyperpolarization induced a slowly activating inwardly rectifying conductance in most protoplasts isolated from the root cortex. In contrast, most protoplasts isolated from the stele contained a slowly activating outwardly rectifying conductance upon plasma membrane depolarization. The reversal potential of the inward current indicated that it was primarily due to the movement of K⁺; the outwardly rectifying conductance was comparatively less selective for K⁺. Membrane hyperpolarization beyond a threshold of about ?70 mV induced inward currents. When E_K was set negative of this threshold, inward currents activated negative of E_K and no outward currents were observed positive of E_K. Outward currents in the stelar protoplasts activated at potentials positive of ?85 mV. However, when E_K was set positive of ?85 mV a small inward current was also observed at potentials negative (and slightly positive) of the equilibrium potential for K⁺. Inwardly and outwardly rectifying K⁺ channels were observed in outside-out patches from the plasma membrane of cortical and stelar cells, respectively. Characterization of these channels showed that they were likely to be responsible for the macroscopic ‘whole-cell’ currents. Inward and outward currents were affected differently by various K⁺ channel blockers (TEA⁺, Ba²⁺ and Cs⁺). In addition, Ca²⁺ above 1 mM partially blocked the inward current in a voltage-dependent manner but had little effect on the outward current. It is suggested that the inwardly rectifying conductance identified in protoplasts isolated from the cortex probably represents an important component of the low-affinity K⁺ uptake mechanism (mechanism II) identified in intact roots. The outwardly rectifying conductance identified in protoplasts isolated from the stele could play a role in the release of cations into the xylem vessels for transport to the shoot.     

Macroscopic Ca2+ -Na+ and K+ currents in single heart and aortic cells

Summary The whole-cell voltage clamp technique was used to study the slow inward currents and K⁺ outward currents in single heart cells of embryonic chick and in rabbit aortic cells. In single heart cells of 3-day-old chick embryo three types of slow inward Na⁺ currents were found. The kinetics and the pharmacology of the slow I_Na, were different from those of the slow Ica in older embryos. Two types of slow inward currents were found in aortic single cells of rabbit; angiotensin 11 increased the sustained type and d-cAMP and d-cGMP decreased the slow transient component. Two types of outward K⁺ currents were found in both aortic and heart cells. Single channel analysis demonstrated the presence of a high single K⁺ channel conductance in aortic cells. In cardiac and vascular smooth muscles, slow inward currents do share some pharmacological properties, although the regulation of these channels by cyclic nucleotides and several drugs seems to be different.     

Towards an understanding of the molecular basis of plants K+ transport: Characterization of cloned K+ transport cDNAs

Recently, two K⁺-transport cDNAs, KAT1 and AKT1, were cloned in Arabidopsis thaliana. These cDNAs had structural similarities to K⁺ channel genes in animals, and also conferred the ability for growth on micromolar levels of K⁺ when expressed in K⁺ transport-defective yeast mutants. In this study, we examined the possibility that KAT1 encodes the high-affinity K⁺ transport system that has been previously characterized in plant roots, by studying the concentration-dependent kinetics of K⁺ transport for KAT1 expressed in Xenopus oocytes and Saccharomyces cerevisiae. In both organisms, the K⁺ transport system encoded by KAT1 yielded Michaelis-Menten kinetics with a high K_m for K⁺ (35 mM in oocytes, 0.6 mM in yeast cells). Furthermore, Northern analysis indicated that KAT1 is expressed primarily in the Arabidopsis shoot. These results strongly suggest that the system encoded by KAT1 is not a root high-affinity K⁺ transporter.     

Modeling squid axon K+ channel by a nucleation and growth kinetic mechanism

A kinetic model accounting for all salient features of the K⁺ channel of the squid giant axon, including the rising phase of the ON gating charge and the Cole-Moore effect, is provided. Upon accounting for a significant feature distinguishing K⁺, Na⁺ and Ca^2 + channels from channel-forming peptides modeled in our previous 2016 BBA paper, the nucleation-and-growth kinetic model developed therein is extended to simulate ON ionic and gating currents of the K⁺ channel of the squid giant axon at different depolarization potentials by the use of only two free parameters. K⁺ channel opening is considered to proceed by progressive aggregation of single subunits, while they are moving their gating charge outward under depolarizing conditions within their tetrameric structure; K⁺ channel closing proceeds in the opposite direction, by repolarization-induced disaggregation of subunits, accompanied by inward movement of their gating charge.