TPK1, a Ca(2+)-regulated Arabidopsis vacuole two-pore K(+) channel is activated by 14-3-3 proteins

The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K(+) channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patch-clamp studies identified TPK1 as a voltage-independent and Ca(2+)-activated K(+) channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca(2+)- and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.     

Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses.

The putative two-pore Ca(2+) channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co-localizes with the K(+)-selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca(2+)-activated slow vacuolar (SV) currents, which were increased in AtTPC1-over-expressing Arabidopsis compared to the wild-type. A Ca(2+)-insensitive vacuolar cation channel, as yet uncharacterized, could be resolved in tpc1-2 knockout plants. The kinetics of ABA- and CO(2)-induced stomatal closure were similar in wild-type and tpc1-2 knockout plants, excluding a role of SV channels in guard-cell signalling in response to these physiological stimuli. ABA-, K(+)-, and Ca(2+)-dependent root growth phenotypes were not changed in tpc1-2 compared to wild-type plants. Given the permeability of SV channels to mono- and divalent cations, the question arises as to whether TPC1 in vivo represents a pathway for Ca(2+) entry into the cytosol. Ca(2+) responses as measured in aequorin-expressing wild-type, tpc1-2 knockout and TPC1-over-expressing plants disprove a contribution of TPC1 to any of the stimulus-induced Ca(2+) signals tested, including abiotic stresses (cold, hyperosmotic, salt and oxidative), elevation in extracellular Ca(2+) concentration and biotic factors (elf18, flg22). In good agreement, stimulus- and Ca(2+)-dependent gene activation was not affected by alterations in TPC1 expression. Together with our finding that the loss of TPC1 did not change the activity of hyperpolarization-activated Ca(2+)-permeable channels in the plasma membrane, we conclude that TPC1, under physiological conditions, functions as a vacuolar cation channel without a major impact on cytosolic Ca(2+) homeostasis.     

Vacuolar ion channel of the yeast, Saccharomyces cerevisiae

Ionic flux is most likely to regulate the chemiosmotic potential differences across vacuolysosomal membranes in animal, plant, and fungal cells. We found a membrane potential-dependent cation channel in yeast vacuolar membrane and characterized its several features by an electrophysiological method using artificial planar bilayer membranes incorporated with isolated yeast vacuolar membrane vesicles. This ion channel conducts K+ (single channel conductance, 435 pS in 0.3 M KCl) and several other monovalent cations (Cs+, Na+, and Li+) with broad selectivity, but does not conduct Cl-. The opening of this channel is regulated by the membrane potential and the presence of calcium ion on the cytoplasmic face. These characteristics suggested that the vacuolar cation channel functions as one of essential components for formation and regulation of the chemical and electrical potential differences across the vacuolar membrane.     

A putative two pore channel AtTPC1 mediates Ca(2+) flux in Arabidopsis leaf cells

The gene encoding voltage-gated channel with high affinity for Ca(2+) permeation has not been cloned from plants. In the present study, we isolated a full-length cDNA encoding a putative Ca(2+ )channel (AtTPC1) from Arabidopsis. AtTPC1 has two conserved homologous domains, both of which contain six transmembrane segments (S1-S6) and a pore loop (P) between S5 and S6 in each domain, and has the highest homology with the two pore channel TPC1 recently cloned from rat. The overall structure is similar to the half of the general structure of alpha-subunits of voltage-activated Ca(2+) channels from animals. AtTPC1 rescued the Ca(2+) uptake activity of a yeast mutant cch1. Sucrose-induced luminescence, which reflects a cytosolic free Ca(2+) increase in aequorin-expressing Arabidopsis leaves, was enhanced by overexpression of AtTPC1 and suppressed by antisense expression of it. Sucrose-H(+) symporters AtSUC1 and 2, which depolarize membrane potential of cells receiving sucrose, also depressed a Ca(2+) increase by their antisense expression. These results suggest that AtTPC1 mediates a voltage-activated Ca(2+ )influx in Arabidopsis leaf cells.     

Potassium-selective channel in the red beet vacuolar membrane

In higher plants the vacuolar K(+)-selective (VK) channel was identified solely in guard cells. This patch-clamp study describes a 40 pS homologue of the VK channel in Beta vulgaris taproot vacuoles. This voltage-independent channel is activated by submicromolar Ca(2+), and is ideally selective for K(+) over Cl(-) and Na(+).     

In planta regulation of the Arabidopsis Ca(2+)/H(+) antiporter CAX1

Vacuolar localized Ca(2+)/H(+) exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca(2+) homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. In yeast expression assays, a 36 amino acid N-terminal truncation of CAX1, termed sCAX1, and variants with specific mutations in this N-terminus, show CAX1-mediated Ca(2+)/H(+) antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca(2+) accumulation and heightened activity of vacuolar Ca(2+)/H(+) antiport. Here the properties of N-terminal CAX1 variants in plants and yeast expression systems are compared and contrasted to determine if autoinhibition of CAX1 is occurring in planta. Initially, using ionome analysis, it has been demonstrated that only yeast cells expressing activated CAX1 transporters have altered total calcium content and fluctuations in zinc and nickel. Tobacco plants expressing activated CAX1 variants displayed hypersensitivity to ion imbalances, increased calcium accumulation, heightened concentrations of other mineral nutrients such as potassium, magnesium and manganese, and increased activity of tonoplast-enriched Ca(2+)/H(+) transport. Despite high in planta gene expression, CAX1 and N-terminal variants of CAX1 which were not active in yeast, displayed none of the aforementioned phenotypes. Although several plant transporters appear to contain N-terminal autoinhibitory domains, this work is the first to document clearly N-terminal-dependent regulation of a Ca(2+) transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits.     

Calcium-Activated K+ Channels and Calcium-Induced Calcium Release by Slow Vacuolar Ion Channels in Guard Cell Vacuoles Implicated in the Control of Stomatal Closure

Stomatal closing requires the efflux of K+ from the large vacuolar organelle into the cytosol and across the plasma membrane of guard cells. More than 90% of the K+ released from guard cells during stomatal closure originates from the guard cell vacuole. However, the corresponding molecular mechanisms for the release of K+ from guard cell vacuoles have remained unknown. Rises in the cytoplasmic Ca2+ concentration have been shown to trigger ion efflux from guard cells, resulting in stomatal closure. Here, we report a novel type of largely voltage-independent K+-selective ion channel in the vacuolar membrane of guard cells that is activated by physiological increases in the cytoplasmic Ca2+ concentration. These vacuolar K+ (VK) channels had a single channel conductance of 70 pS with 100 mM KCI on both sides of the membrane and were highly selective for K+ over NH4+ and Rb+. Na+, Li+, and Cs+ were not measurably permeant. The Ca2+, voltage, and pH dependences, high selectivity for K+, and high density of VK channels in the vacuolar membrane of guard cells suggest a central role for these K+ channels in the initiation and control of K+ release from the vacuole to the cytoplasm required for stomatal closure. The activation of K+-selective VK channels can shift the vacuolar membrane to more positive potentials on the cytoplasmic side, sufficient to activate previously described slow vacuolar cation channels (SV-type). Analysis of the ionic selectivity of SV channels demonstrated a Ca2+ over K+ selectivity (permeability ratio for Ca2+ to K+ of ~3:1) of these channels in broad bean guard cells and red beet vacuoles, suggesting that SV channels play an important role in Ca2+-induced Ca2+ release from the vacuole during stomatal closure. A model is presented suggesting that the interaction of VK and SV channel activities is crucial in regulating vacuolar K+ and Ca2+ release during stomatal closure. Furthermore, the possibility that the ubiquitous SV channels may represent a general mechanism for Ca2+-induced Ca2+ release from higher plant vacuoles is discussed.     

Characterization of a tobacco TPK-type K+ channel as a novel tonoplast K+ channel using yeast tonoplasts

The tonoplast K(+) membrane transport system plays a crucial role in maintaining K(+) homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K(+) channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased approximately 2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K(+) over Na(+). NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca(2+), but a cytosolic concentration of 45 microM Ca(2+) resulted in a 2-fold increase in the amplitude of the K(+) current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K(+) currents. These results show that NtTPK1 is a novel tonoplast K(+) channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.     

Vacuolar two-pore K+ channels act as vacuolar osmosensors

? Plant two-pore K(+) channels (TPKs) have been shown previously to play a role in vacuolar K(+) homeostasis. TPK activity is insensitive to membrane voltage, but regulated by cytoplasmic calcium and 14-3-3 proteins. This study reports that membrane stretch and osmotic gradients also alter the activity of TPKs from Arabidopsis, rice and barley, and that this may have a physiological relevance for osmotic homeostasis. ? Mechanosensitivity was studied using patch clamp experiments and TPKs from Arabidopsis, rice and barley. In addition, the capability of TPKs to act as osmosensors was determined. By using protoplast disruption assays and intact plant survival assays, in genotypes that differed in TPK expression, the physiological relevance of TPK-based osmosensing was tested. ? TPKs from all three species showed varying degrees of mechanosensitivity. TPK activity in channels from all three species was sensitive to trans-tonoplast osmotic gradients. TPK osmosensing is likely to proceed via the detection of small perturbations in membrane tension. Intact plant and protoplast assays showed that TPK-based osmosensing is important during exposure to rapid changes in external osmolarity. ? Vacuolar TPK channels can act as intracellular osmosensors and rapidly increase channel activity during hypo-osmotic shock to release vacuolar K(+) .     

A type of voltage-dependent Ca2+ channel on Vicia faba guard cell plasma membrane outwardly permeates K+

The fine regulation of stomatal aperture is important for both plant photosynthesis and transpiration, while stomatal closing is an essential plant response to biotic and abiotic stresses such as drought, salinity, wounding, and pathogens. Quick stomatal closing is primarily due to rapid solute loss. Cytosolic free calcium ([Ca(2+)](cyt)) is a ubiquitous second messenger, and its elevation or oscillation plays important roles in stomatal movements, which can be triggered by the opening of Ca(2+)-permeable channels on the plasma membrane. For Ca(2+)-permeable channel recordings, Ba(2+) is preferred as a charge-carrying ion because it has higher permeability to Ca(2+) channels and blocks K(+) channel activities to facilitate current recordings; however, it prevents visualization of Ca(2+) channels' K(+) permeability. Here, we employed Ca(2+) instead of Ba(2+) in recording Ca(2+)-permeable channels on Vicia faba guard cell plasma membrane to mimic physiological solute conditions inside guard cells more accurately. Inward Ca(2+) currents could be recorded at the single-channel level, and these currents could be inhibited by micromolar Gd(3+), but their reversal potential is far away from the theoretical equilibrium potential for Ca(2+). Further experiments showed that the discrepancy of the reversal potential of the recorded Ca(2+) currents is influenced by cytosolic K(+). This suggests that voltage-dependent Ca(2+) channels also mediate K(+) efflux at depolarization voltages. In addition, a new kind of high-conductance channels with fivefold to normal Ca(2+) channel and 18-fold to normal outward K(+) conductance was found. Our data presented here suggest that plants have their own saving strategies in their rapid response to stress stimuli, and multiple kinds of hyperpolarization-activated Ca(2+)-permeable channels coexist on plasma membranes.     

New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency.

Potassium (K+) channels mediating important physiological functions are characterized by a common pore-forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of ''two-pore'' K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium-binding EF-hand motifs. Heterologous expression of KCO1 in baculovirus-infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+-selective currents elicited by depolarizing voltage pulses in whole-cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to <150 nM. However, KCO1 strongly activated at increasing [Ca2+]cyt, with a saturating activity observed at approximately 300 nM [Ca2+]cyt. KCO1 single channel analysis on excised membrane patches, resulting in a single channel conductance of 64 pS, confirmed outward rectification as well as Ca2+-dependent activation. These data suggest a direct link between calcium-mediated signaling processes and K+ ion transport in higher plants. The identification of KCO1 as the first plant K+ outward channel opens a new field of structure-function studies in plant ion channels.     

A novel motif essential for SNARE interaction with the K(+) channel KC1 and channel gating in Arabidopsis

The SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane, and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K(+) channel subunit KC1 and forms a tripartite complex with a second K(+) channel subunit, AKT1, to control channel gating and K(+) transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for protein-protein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this motif is the minimal requirement for SNARE-dependent alterations in K(+) channel gating when heterologously expressed, and that rescue of KC1-associated K(+) current of the root epidermis in syp121 mutant Arabidopsis plants depends on expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously unidentified motif required for SNARE-ion channel interactions and lead us to suggest a mechanistic framework for understanding the coordination of vesicle traffic with transmembrane ion transport.     

Further characterization of the cation channel of a yeast vacuolar membrane in a planar lipid bilayer

A voltage-dependent and Ca2(+)-activated cation channel recently found in the vacuolar membrane of the yeast Saccharomyces cerevisiae was incorporated into planar lipid bilayers and further characterized in macroscopic and single channel levels. Single channel conductances for various cations were in the order: NH4+ greater than K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+, and were nearly consistent with the order of permeability ratio estimated from reversal potentials determined by macroscopic measurement. Up to 6 mM of Ca2+ added to the cis (cytoplasmic) side opened the channel, but higher concentrations closed the channel without affecting the single channel conductance. Ba2+ closed the channel without affecting the single channel conductance. Ba2+ closed the channel from the cis side. In addition to the above channel, a small cation-selective channel of about 40 pS was found.     

Homeostatic control of slow vacuolar channels by luminal cations and evaluation of the channel-mediated tonoplast Ca2+ fluxes in situ

Ca(2+), Mg(2+), and K(+) activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca(2+), by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca(2+) activity was approximately 0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca(2+) in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca(2+)) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca(2+) within a physiological range of concentrations (0.1-1.0 mM). Aggregation of the physiological vacuolar Na(+) (60 mM) and Mg(2+) (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca(2+) variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca(2+) activates the SV channel in a voltage-independent manner with K(d)=0.7-1.5 microM. Comparison of the vacuolar Ca(2+) fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 microM) cytosolic Ca(2+), only 0.5% of SV channels are open. This mediates a Ca(2+) release of only a few pA per vacuole (approximately 0.1 pA per single SV channel). Overall, our data suggest that the release of Ca(2+) through SV channels makes little contribution to a global cytosolic Ca(2+) signal.     

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

Characterization of plant phenotypes associated with loss-of-function of AtCNGC1, a plant cyclic nucleotide gated cation channel.

Of the 57 cation channel genes in the Arabidopsis genome, over a third encode cyclic nucleotide gated cation channels (CNGCs). CNGCs are ion channels regulated by cytosolic signaling molecules (cyclic nucleotides, calmodulin, and Ca(2+)), and which conduct Ca(2+) as well as K(+) and in some cases Na(+). Little is currently known about the role CNGCs may play in plant growth and development. Here, we examined the hypothesis that an Arabidopsis thaliana genotype containing a null mutation in one of the CGNC genes (AtCNGC1) would display cation uptake-related growth phenotype differences from wild type (WT) plants. We determined that AtCNGC1 protein is primarily expressed in the roots of Arabidopsis seedlings. Seedlings lacking this protein had slightly (6-22%) lower shoot Ca(2+) than WT plants. Primary roots of Atcngc1 mutant seedlings grew faster than roots of WT plants, and had larger angles of gravicurvature and less nitric oxide generation upon gravistimulation. We conclude that channels formed (at least in part) by AtCNGC1 contribute (along with other channels) to Ca(2+) uptake into plants, and that Ca(2+) uptake into roots through AtCNGC1 affects some aspects of growth in the primary root of Arabidopsis seedlings.     

Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca(2+) is required for both structural and biophysical roles. In addition, changes in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) orchestrate responses to developmental and environmental signals. In many instances, [Ca(2+)](cyt) is increased by Ca(2+) influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca(2+)-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca(2+)-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca(2+)-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca(2+) channels, the genetic counterparts of specific Ca(2+)-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca(2+) channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca(2+) channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La(3+) and nifedipine, and their increased activity as [Ca(2+)](cyt) is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca(2+)-permeable) K(+) channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.     

Ion conductance pathways in potato tuber (Solanum tuberosum) inner mitochondrial membrane

Single-ion channel activities were measured after reconstitution of potato tuber mitochondrial inner membranes into planar lipid bilayers. In addition to the recently described large-conductance Ca(2+)-activated potassium channel activity (Koszela-Piotrowska et al., 2009), the following mitochondrial ion conductance pathways were recorded: (i) an ATP-regulated potassium channel (mitoK(ATP) channel) activity with a conductance of 164+/-8pS, (ii) a large-conductance Ca(2+)-insensitive iberiotoxin-sensitive potassium channel activity with a conductance of 312 pS+/-23, and (iii) a chloride 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-inhibited channel activity with a conductance of 117 pS+/-4. In isolated non-phosphorylating potato tuber mitochondria, individual and combined potassium channel activities caused significant (up to 14mV) but not collapsing K(+)-influx-induced membrane potential depolarisation. Under phosphorylating conditions, the coupling parameters were unchanged in the presence of high K(+) level, indicating that plant K(+) channels function as energy-dissipating systems that are not able to divert energy from oxidative phosphorylation. A potato tuber K(+) channel that is ATP-, 5-hydroxydecanonic acid-, glybenclamide-inhibited and diazoxide-stimulated caused low cation flux, modestly decreasing membrane potential (up to a few mV) and increasing respiration in non-phosphorylating mitochondria. Immunological analysis with antibodies raised against the mammalian plasma membrane ATP-regulated K(+) channel identified a pore-forming subunit of the Kir-like family in potato tuber mitochondrial inner membrane. These results suggest that a mitoK(ATP) channel similar to that of mammalian mitochondria is present in potato tuber mitochondria.     

Internal Ca(2+) release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue.

Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals. In mammalian cells, the release of Ca(2+) from intracellular compartments is required for signaling and involves the regulated opening of ryanodine and inositol-1,4,5-trisphosphate (IP3) receptors. However, in budding yeast, no signaling pathway has been shown to involve Ca(2+) release from internal stores, and no homologues of ryanodine or IP3 receptors exist in the genome. Here we show that hyperosmotic shock provokes a transient increase in cytosolic Ca(2+) in vivo. Vacuolar Ca(2+), which is the major intracellular Ca(2+) store in yeast, is required for this response, whereas extracellular Ca(2+) is not. We aimed to identify the channel responsible for this regulated vacuolar Ca(2+) release. Here we report that Yvc1p, a vacuolar membrane protein with homology to transient receptor potential (TRP) channels, mediates the hyperosmolarity induced Ca(2+) release. After this release, low cytosolic Ca(2+) is restored and vacuolar Ca(2+) is replenished through the activity of Vcx1p, a Ca(2+)/H(+) exchanger. These studies reveal a novel mechanism of internal Ca(2+) release and establish a new function for TRP channels.