Channel KAT1 Research Articles

Rice Shaker Potassium Channel OsKAT1 Confers Tolerance to Salinity Stress on Yeast and Rice Cells

We screened a rice (Oryza sativa L. 'Nipponbare') full-length cDNA expression library through functional complementation in yeast (Saccharomyces cerevisiae) to find novel cation transporters involved in salt tolerance. We found that expression of a cDNA clone, encoding the rice homolog of Shaker family K(+) channel KAT1 (OsKAT1), suppressed the salt-sensitive phenotype of yeast strain G19 (Deltaena1-4), which lacks a major component of Na(+) efflux. It also suppressed a K(+)-transport-defective phenotype of yeast strain CY162 (Deltatrk1Deltatrk2), suggesting the enhancement of K(+) uptake by OsKAT1. By the expression of OsKAT1, the K(+) contents of salt-stressed G19 cells increased during the exponential growth phase. At the linear phase, however, OsKAT1-expressing G19 cells accumulated less Na(+) than nonexpressing cells, but almost the same K(+). The cellular Na(+) to K(+) ratio of OsKAT1-expressing G19 cells remained lower than nonexpressing cells under saline conditions. Rice cells overexpressing OsKAT1 also showed enhanced salt tolerance and increased cellular K(+) content. These functions of OsKAT1 are likely to be common among Shaker K(+) channels because OsAKT1 and Arabidopsis (Arabidopsis thaliana) KAT1 were able to complement the salt-sensitive phenotype of G19 as well as OsKAT1. The expression of OsKAT1 was restricted to internodes and rachides of wild-type rice, whereas other Shaker family genes were expressed in various organs. These results suggest that OsKAT1 is involved in salt tolerance of rice in cooperation with other K(+) channels by participating in maintenance of cytosolic cation homeostasis during salt stress and thus protects cells from Na(+).

From sequence to antibody: Genetic immunisation is suitable to generate antibodies against a rare plant membrane protein, the KAT 1 channel

Monoclonal antibodies against the K + channel KAT1 of Arabidopsis thaliana, a low abundance, plant plasma membrane protein, were generated by genetic immunisation to avoid the time and labour consuming purification of native or recombinant proteins and peptides usually necessary for conventional immunisation techniques. The resulting polyclonal and monoclonal antibody sera recognised a single protein band in a microsomal fraction of wild-type A. thaliana leaves and in membrane fractions of transgenic yeast cells and tobacco plants expressing the KAT1 protein. Therefore, genetic immunisation is suitable for generating monoclonal antibodies against plant proteins and particularly, against plant membrane proteins of low abundance.

Structure prediction for the down state of a potassium channel voltage sensor

Voltage-gated potassium (Kv) channels, essential for regulating potassium uptake and cell volume in plants and electrical excitability in animals, switch between conducting and non-conducting states as a result of conformational changes in the four voltage-sensing domains (VSDs) that surround the channel pore. This process, known as gating, is initiated by a cluster of positively charged residues on the fourth transmembrane segment (S4) of each VSD, which drives the VSD into a 'down state' at negative voltages and an 'up state' at more positive voltages. The crystal structure of Kv1.2 probably corresponds to the up state, but the local environment of S4 in the down state and its motion in voltage gating remains unresolved. Here we employed several conditional lethal/second-site suppressor yeast screens to determine the transmembrane packing of the VSD in the down state. This screen relies on the ability of KAT1, a eukaryotic Kv channel, to conduct potassium when its VSDs are in the down state, thereby rescuing potassium-transport-deficient yeast. Starting with KAT1 channels bearing conditional lethal mutations, we identified second-site suppressor mutations throughout the VSD that recover yeast growth. We then constructed a down state model of the channel using six pairs of interacting residues as structural constraints and verified this model by engineering suppressor mutations on the basis of spatial considerations. A comparison of this down state model with the up state Kv1.2 structure suggests that the VSDs undergo large rearrangements during gating, whereas the S4 segment remains positioned between the central pore and the remainder of the VSD in both states.

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.

Diacidic Motif Is Required for Efficient Transport of the K+ Channel KAT1 to the Plasma Membrane

For a number of mammalian ion channels, trafficking to the plasma membrane was found to be controlled by intrinsic sequence motifs. Among these sequences are diacidic motifs that function as endoplasmic reticulum (ER) export signals. So far it is unclear if similar motifs also exist in plant ion channels. In this study we analyzed the function of four diacidic DXE/DXD motifs of the plant K(+) channel KAT1. Mutation of the first diacidic DXE motif resulted in a strong reduction of the KAT1 conductance in both guard cell protoplasts and HEK293 cells (human embryonic kidney cells). Confocal fluorescence microscopy of guard cells expressing the mutated KAT1 fused to green fluorescent protein revealed localization of the mutated channel only in intracellular structures around the nucleus. These structures could be identified as part of the ER via coexpression of KAT1 fused to yellow fluorescent protein with an ER-retained protein (HDEL) fused to cyan fluorescent protein. Block of vesicle formation from the ER by overexpression of the small GTP-binding protein Sar1 fixed in its GDP-bound form led to retention of wild-type KAT1 in similar parts of the ER. Mutation of the three other diacidic motifs had no effect. Together, the results demonstrate that one diacidic motif of KAT1 is essential for ER export of the functional channel in both guard cell protoplasts and HEK293 cells. This suggests that trafficking of plant plasma membrane ion channels is controlled via a conserved mechanism.

A Unique Voltage Sensor Sensitizes the Potassium Channel AKT2 to Phosphoregulation

Among all voltage-gated K+ channels from the model plant Arabidopsis thaliana, the weakly rectifying K+ channel (Kweak channel) AKT2 displays unique gating properties. AKT2 is exceptionally regulated by phosphorylation: when nonphosphorylated AKT2 behaves as an inward-rectifying potassium channel; phosphorylation of AKT2 abolishes inward rectification by shifting its activation threshold far positive (>200 mV) so that it closes only at voltages positive of +100 mV. In its phosphorylated form, AKT2 is thus locked in the open state in the entire physiological voltage range. To understand the molecular grounds of this unique gating behavior, we generated chimeras between AKT2 and the conventional inward-rectifying channel KAT1. The transfer of the pore from KAT1 to AKT2 altered the permeation properties of the channel. However, the gating properties were unaffected, suggesting that the pore region of AKT2 is not responsible for the unique Kweak gating. Instead, a lysine residue in S4, highly conserved among all Kweak channels but absent from other plant K+ channels, was pinpointed in a site-directed mutagenesis approach. Substitution of the lysine by serine or aspartate abolished the “open-lock” characteristic and converted AKT2 into an inward-rectifying channel. Interestingly, phosphoregulation of the mutant AKT2-K197S appeared to be similar to that of the Kin channel KAT1: as suggested by mimicking the phosphorylated and dephosphorylated states, phosphorylation induced a shift of the activation threshold of AKT2-K197S by about +50 mV. We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation. The phosphorylation-induced reduction of the activation energy in AKT2 is ∼6 kT larger than in the K197S mutant. It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it.

The S4 Voltage Sensor Packs Against the Pore Domain in the KAT1 Voltage-Gated Potassium Channel

In voltage-gated ion channels, the S4 transmembrane segment responds to changes in membrane potential and controls channel opening. The local environment of S4 is still unknown, even regarding the basic question as to whether S4 is close to the pore domain. Relying on the ability of functional KAT1 channels to rescue potassium (K+) transport-deficient yeast, we have performed an unbiased mutagenesis screen aimed at determining whether S4 packs against S5 of the pore domain. Starting with semilethal mutations of surface-exposed S5 residues of the KAT1 pore domain, we have screened randomly mutagenized libraries of S4 or S1-S3 for second-site suppressors. Our study identifies two S4 residues that interact in a highly specific manner with two S5 residues in the middle of the membrane-spanning regions, supporting a model in which the S4 voltage sensor packs against the pore domain in the hyperpolarized, or "down," state of S4.

Plant K in and K out channels: Approaching the trait of opposite rectification by analyzing more than 250 KAT1–SKOR chimeras

Members of the Shaker-like plant K + channel family share a common structure, but are highly diverse in their function: they behave as either hyperpolarization-activated inward-rectifying (K in) channels, or leak-like (K weak) channels, or depolarization-activated outward-rectifying (K out) channels. Here we created 256 chimeras between the K in channel KAT1 and the K out channel SKOR. The chimeras were screened in a potassium-uptake deficient yeast strain to identify those, which mediate potassium inward currents, i.e., which are functionally equivalent to KAT1. This strategy allowed us to identify three chimeras which differ from KAT1 in three parts of the polypeptide: the cytosolic N-terminus, the cytosolic C-terminus, and the putative voltage-sensor S4. Additionally, mutations in the K out channel SKOR were generated in order to localize molecular entities underlying its depolarization activation. The triple mutant SKOR-D312N-M313L-I314G, carrying amino-acid changes in the S6 segment, was identified as a channel which did not display any rectification in the tested voltage-range.

Orientation of Arabidopsis thaliana 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 NH(2) 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.

K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions.

Organization of proteins into complexes is crucial for many cellular functions. However, most proteomic approaches primarily detect protein interactions for soluble proteins but are less suitable for membrane-associated complexes. Here we describe a mating-based split ubiquitin system (mbSUS) for systematic identification of interactions between membrane proteins as well as between membrane and soluble proteins. mbSUS allows in vivo cloning of PCR products into a vector set, detection of interactions via mating, regulated expression of baits, and improved selection of interacting proteins. Cloning is simplified by introduction of lambda attachment sites for GATEWAY. Homo- and heteromeric interactions between Arabidopsis K(+) channels KAT1, AKT1, and AKT2 were identified. Tests with deletion mutants demonstrate that the C terminus of KAT1 and AKT1 is necessary for physical assembly of complexes. Screening of a sorted collection of 84 plant proteins with K(+) channels as bait revealed differences in oligomerization between KAT1, AKT1, and AtKC1, and allowed detection of putative interacting partners of KAT1 and AtKC1. These results show that mbSUS is suited for systematic analysis of membrane protein interactions.

Endocytosis against high turgor: intact guard cells of Vicia faba constitutively endocytose fluorescently labelled plasma membrane and GFP-tagged K-channel KAT1.

The relevance of endocytosis in plants against high turgor pressure has frequently been questioned on the basis of energetic considerations. Here, we examine the dynamics of the plasma membrane (PM) in turgid guard cells of Vicia faba by monitoring with confocal microscopy the fate of fluorescent styryl dyes (FM1-43, FM2-10 and FM4-64). As a second marker, we also observe the retrieval of a fluorescent chimaera of the K(+)-inward rectifying channel from Arabidopsis thaliana and the green fluorescent protein (KAT1::GFP). Analysis of cytoplasmic structures, which became labelled by the different styryl dyes, revealed that only FM4-64, the most hydrophobic dye, was a reliable marker of endocytosis, whereas the two other styryl dyes resulted also in an unspecific labelling of different cytoplasmic structures including mitochondria. Over some minutes of incubation in continuous presence of these dyes, endocytic vesicles in the cortical cytoplasm beneath the PM were fluorescently labelled. The identification is based on the observation that the size distribution of these structures is very similar to that of endocytic vesicles obtained from patch-clamp capacitance recordings. Also, these structures are frequently co-labelled with KAT1::GFP. Taken together, the data show that turgid guard cells undergo vigorous constitutive endocytosis and retrieve membrane including the K(+)-channel KAT1 from the PM via endocytic vesicles.

Yeast hygromycin sensitivity as a functional assay of cyclic nucleotide gated cation channels

Cyclic nucleotide gated cation channels (CNGCs) are a large (20 genes in Arabidopsis thaliana) family of plant ligand gated (i.e. cyclic nucleotides activate currents) ion channels, however, little is known about their functional properties. One reason for this is the recalcitrance of plant CNGC expression in heterologous systems amenable to patch clamp studies. Here, we show results demonstrating the efficacy of using growth of a K + uptake-deficient yeast ( trk1,2) as a functional assay of CNGCs as inwardly-conducting cell membrane cation (K +) transporters. Prior work demonstrated that trk1,2 is hypersensitive to the antibiotic hygromycin (hyg) and that expression of an inwardly conducting K + transporter suppresses hyg hypersensitivity. We find that increasing [hyg] in solid YPD medium inhibits trk1,2 growth around a filter disk saturated with 3 M K +. Northern analysis indicated that message is transcribed in trk1,2 transformed with the CNGC coding sequences. Confocal imaging of yeast expressing CNGC-fluorescent fusion proteins indicated channel targeting to the cell membrane. Trk1,2 expressing several plant CNGCs grown in the presence of hyg demonstrated (a) greater growth than trk1,2 transformed with empty plasmid, and (b) enhanced growth when cAMP was added to the medium. Alternatively, cAMP inhibited growth of yeast transformed with either the empty plasmid, or the plant K + channel KAT1; this channel is not a CNGC. Growth of trk1,2 was dependent on filter disk [K +]; suggesting that complementation of hyg hypersensitivity due to presence of a functional plant CNGC was dependent on K + movement into the cytosol. We conclude that plant CNGC functional characterization can be facilitated by this assay system.

Trafficking of the plant potassium inward rectifier KAT1 in guard cell protoplasts of Vicia faba.

Trafficking of K+ inward (Kin+) rectifying channels was analyzed in guard cells of Vicia faba transfected with the Kin+ rectifier from Arabidopsis thaliana KAT1 fused to the green fluorescent protein (GFP). Confocal images and whole-cell patch-clamp measurements confirmed the incorporation of active KAT1 channels into the plasma membrane of transfected guard cell protoplasts. The Kin+ rectifier current density of the plasma membrane was much larger in transfected protoplasts than in wild-type (wt) protoplasts. This shows a coupling between K+ channel synthesis and incorporation of the channel into the plasma membrane. Pressure-driven increase and decrease in surface area led to the incorporation and removal of vesicular membrane carrying active Kin+ rectifier in wt and transfected protoplasts. These vesicular membranes revealed a higher channel density than the plasma membrane, suggesting that Kin+ rectifier remains in clusters during trafficking to and from the plasma membrane. The observed results can be explained by a model illustrating that vesicles of a pre-plasma membrane pool carry K+ channels preferentially in clusters during constitutive and pressure-driven exo- and endocytosis.

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 Zn 2+. 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 Pb 2+ and Cd 2+ and inhibited by Ni 2+. 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 Pb 2+ or Cd 2+. These results suggest that Zn 2+ 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.

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 (τ = 318 ± 34 μs 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 (≈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.

The K+ Channel KZM1 Mediates Potassium Uptake into the Phloem and Guard Cells of the C4 Grass Zea mays

In search of K(+) channel genes expressed in the leaf of the C(4) plant Zea mays, we isolated the cDNA of KZM1 (for K(+) channel Zea mays 1). KZM1 showed highest similarity to the Arabidopsis K(+) channels KAT1 and KAT2, which are localized in guard cells and phloem. When expressed in Xenopus oocytes, KZM1 exhibited the characteristic features of an inward-rectifying, potassium-selective channel. In contrast to KAT1- and KAT2-type K(+) channels, however, KZM1 currents were insensitive to external pH changes. Northern blot analyses identified the leaf, nodes, and silks as sites of KZM1 expression. Following the separation of maize leaves into epidermal, mesophyll, and vascular fractions, quantitative real-time reverse transcriptase-PCR allowed us to localize KZM1 transcripts predominantly in vascular strands and the epidermis. Cell tissue separation and KZM1 localization were followed with marker genes such as the bundle sheath-specific ribulose-1,5-bisphosphate carboxylase, the phloem K(+) channel ZMK2, and the putative sucrose transporter ZmSUT1. When expressed in Xenopus oocytes, ZmSUT1 mediated proton-coupled sucrose symport. Coexpression of ZmSUT1 with the phloem K(+) channels KZM1 and ZMK2 revealed that ZMK2 is able to stabilize the membrane potential during phloem loading/unloading processes and KZM1 to mediate K(+) uptake. During leaf development, sink-source transitions, and diurnal changes, KZM1 is constitutively expressed, pointing to a housekeeping function of this channel in K(+) homeostasis of the maize leaf. Therefore, the voltage-dependent K(+)-uptake channel KZM1 seems to mediate K(+) retrieval and K(+) loading into the phloem as well as K(+)-dependent stomatal opening.

Structure and function of potassium channels in plants: some inferences about the molecular origin of inward rectification in KAT1 channels (Review).

Potassium channels in plants play a variety of important physiological roles including K(+) uptake into roots, stomatal and leaf movements, and release of K(+) into the xylem. This review summarizes current knowledge about a class of plant genes whose products are K(+) channel-forming proteins. Potassium channels of this class belong to a superfamily characterized by six membrane-spanning domains (S1-6), a positively charged S4 domain and a region between the S5 and S6 segments that forms the channel selectivity filter. These channels are voltage dependent, which means the membrane potential modifies the probability of opening (P(o)). However, despite these channels sharing the same topology as the outward-rectifying K(+) channels, which are activated by membrane depolarization, some plant K(+) channels such as KAT1/2 and KST1 open with hyperpolarizing voltages. In outward-rectifying K(+) channels, the change in P(o) is achieved through a voltage sensor formed by the S4 segment that detects the voltage transferring its energy to the gate that controls pore opening. This coupling is achieved by an outward displacement of the charges contained in S4. In KAT1, most of the results indicate that S4 is the voltage sensor. However, how the movement of S4 leads to opening remains unanswered. On the basis of recent data, we propose here that in plant-inward rectifiers an inward movement of S4 leads to channel opening and that the difference between it and outward-rectifying channels resides in the mechanism that couples gating charge displacement with pore opening.

Sodium blocking induced by a point mutation at the C-terminal end of the pore helix of the KAT1 channel.

A plant hyperpolarization-activating K+ channel, KAT1, is highly selective for K+ over Na+ and is little affected by external Na+, which is crucial to take up K+ effectively in a Na+-containing environment. It has been shown that a mutation at the location (Thr256) preceding the selectivity signature sequence dramatically enhanced the sensitivity of the KAT1 channel to external Na+. We report here electrophysiological experiments for the mechanism of action of external Na+ on KAT1 channels. The Thr256 residue was substituted with either glutamine (Q) or glutamate (E). The wild-type channel was insensitive to external Na+. However, the activity of both mutant channels was significantly depressed by Na+ with apparent dissociation constants of 6.7 mm and 11.3 mm for T256Q and T256E, respectively. The instantaneous current-voltage relationships revealed distinct blocking mechanisms for these mutants. For T256Q a typical voltage-dependent fast blocking was shown. On the other hand, the blocking for the T256E mutant was voltage-independent at low Na+ concentrations and became voltage-dependent at higher concentrations. At extreme hyperpolarization the blocking was relieved significantly. These data strongly suggest that the mutation at the end of the pore helix rearranged the selectivity filter and allows Na+ to penetrate into the pore.

Internal aluminum block of plant inward K(+) channels.

Aluminum (Al) inhibits inward K(+) channels (K(in)) in both root hair and guard cells, which accounts for at least part of the Al toxicity in plants. To understand the mechanism of Al-induced K(in) inhibition, we performed patch clamp analyses on K(in) in guard cells and on KAT1 channels expressed in Xenopus oocytes. Our results show that Al inhibits plant K(in) by blocking the channels at the cytoplasmic side of the plasma membrane. In guard cells, single-channel recording revealed that Al inhibition of K(in) occurred only upon internal exposure. Using both "giant patch" recording and single-channel analyses, we found that Al reduced KAT1 open probability and changed its activation kinetics through an internal membrane-delimited mechanism. We also provide evidence that a Ca(2)+ channel-like pathway that is sensitive to antagonists verapamil and La(3)+ mediates Al entry across the plasma membrane. We conclude that Al enters plant cells through a Ca(2)+ channel-like pathway and inhibits K(+) uptake by internally blocking K(in).

Internal Aluminum Block of Plant Inward K+ Channels

Aluminum (Al) inhibits inward K(+) channels (K(in)) in both root hair and guard cells, which accounts for at least part of the Al toxicity in plants. To understand the mechanism of Al-induced K(in) inhibition, we performed clamp analyses on K(in) in guard cells and on KAT1 channels expressed in Xenopus oocytes. Our results show that Al inhibits plant K(in) by blocking the channels at the cytoplasmic side of the plasma membrane. In guard cells, single-channel recording revealed that Al inhibition of K(in) occurred only upon internal exposure. Using both giant patch recording and single-channel analyses, we found that Al reduced KAT1 open probability and changed its activation kinetics through an internal membrane-delimited mechanism. We also provide evidence that a Ca(2)+ channel-like pathway that is sensitive to antagonists verapamil and La(3)+ mediates Al entry across the plasma membrane. We conclude that Al enters plant cells through a Ca(2)+ channel-like pathway and inhibits K(+) uptake by internally blocking K(in).