Anion Currents Research Articles

H2O2 Stimulates Cystic Fibrosis Transmembrane Conductance Regulator through an Autocrine Prostaglandin Pathway, Using Multidrug-Resistant Protein–4

Cystic fibrosis transmembrane conductance regulator (CFTR) activity is essential for the maintenance of airway surface liquid depth, and therefore mucociliary clearance. Reactive oxygen species, increased during inflammatory airway diseases, alter CFTR activity. Here, H2O2 levels in the surface liquid of normal human bronchial epithelial cultures differentiated at the air-liquid interface were estimated, and H2O2-mediated changes in CFTR activity were examined. In Ussing chambers, H2O2-induced anion currents were sensitive to the CFTR inhibitors CFTRinh172 and GlyH-101. These currents were absent in cells from patients with cystic fibrosis. Responses to greater than 500 μM H2O2 were transient. Cyclooxygenase inhibitors blocked the H2O2 response, as did EP1 and EP4 receptor antagonists. A multidrug-resistant protein (MRP) inhibitor and short hairpin RNA directed against MRP4 blocked H2O2 responses. EP1 and EP4 agonists mimicked H2O2 in both control and MRP4 knockdown cells. Thus, H2O2 activates the synthesis, export, and binding of prostanoids via EP4 and, interestingly, EP1 receptors in normal, differentiated human airway epithelial cells to activate cyclic adenosine monophosphate pathways that in turn activate CFTR channels in the apical membrane.

Open Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsis guard cells

Open Stomata 1 (OST1) (SnRK2.6 or SRK2E), a serine/threonine protein kinase, is a positive regulator in abscisic acid (ABA)-mediated stomatal response, but OST1-regulation of K(+) and Ca(2+) currents has not been studied directly in guard cells and it is unknown whether OST1 activity is limiting in ABA-mediated stomatal responses. We employed loss-of-function and gain-of-function approaches to study native ABA responses of Arabidopsis guard cells. We performed stomatal aperture bioassays, patch clamp analyses and reactive oxygen species (ROS) measurements. ABA inhibition of inward K(+) channels and light-induced stomatal opening are reduced in ost1 mutants while transgenic plants overexpressing OST1 show ABA hypersensitivity in these responses. ost1 mutants are insensitive to ABA-induced stomatal closure, regulation of slow anion currents, Ca(2+) -permeable channel activation and ROS production while OST1 overexpressing lines are hypersensitive for these responses, resulting in accelerated stomatal closure in response to ABA. Overexpression of OST1 in planta in the absence of ABA application does not affect basal apertures or ion currents. Moreover, we demonstrate the physical interaction of OST1 with the inward K(+) channel KAT1, the anion channel SLAC1, and the NADPH oxidases AtrbohD and AtrbohF. Our findings support OST1 as a critical limiting component in ABA regulation of stomatal apertures, ion channels and NADPH oxidases in Arabidopsis guard cells.

Regulation of ANO1/TMEM16A anion permeability

Background/aim: Bicarbonate (HCO3 ) is an important ingredient in many biological fluids including saliva. However, calcium activated chlo ride channels (CaCCs) in salivary gland acinar cells are known to be poorly permeable to HCO3 in most cases. Methods: To elucidate the possibility of ANO1 as a HCO3 channel, anion currents were measured in HEK 293T cells transfected with human ANO1 (ac isoform) using the whole cell patch clamp techniques. Results: Relative permeabilities of HCO3 and various other anions were altered by high cytosolic calcium signals inwhole cell patch clamp. Further studies including biochemical and excised patch experiments revealed that a direct association between ANO1 and calmodulin at high [Ca2þ]i and an increase in the dielectric constant of ANO1 anion selectivity filter are responsible for changes in anion permeability. In addition, the high [Ca2þ]i induced increase in HCO3 permeability was reproduced in mouse sub mandibular gland acinar cells, in which ANO1 plays a critical role in fluid secretion. Conclusions: Anion selectivity in ANO1 is dynamically regulated by Ca2þ/calmodulin.

Insights into MLC pathogenesis: GlialCAM is an MLC1 chaperone required for proper activation of volume-regulated anion currents

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes and is associated with myelin and astrocyte vacuolation. It has been suggested that MLC is caused by impaired cell volume regulation as a result of defective activation of astrocytic volume-regulated anion currents (VRAC). GlialCAM brings MLC1 and the ClC-2 Cl(-) channel to cell-cell junctions, even though the role of ClC-2 in MLC disease remains incompletely understood. To gain insights into the biological role of GlialCAM in the pathogenesis of MLC disease, here we analyzed the gain- and loss-of-function phenotypes of GlialCAM in Hela cells and primary astrocytes, focusing on its interaction with the MLC1 protein. Unexpectedly, GlialCAM ablation provoked intracellular accumulation and reduced expression of MLC1 at the plasma membrane. Conversely, over-expression of GlialCAM increased the cellular stability of mutant MLC1 variants. Reduction in GlialCAM expression resulted in defective activation of VRAC and augmented vacuolation, phenocopying MLC1 mutations. Importantly, over-expression of GlialCAM together with MLC1 containing MLC-related mutations was able to reactivate VRAC currents and to reverse the vacuolation caused in the presence of mutant MLC1. These results indicate a previously unrecognized role of GlialCAM in facilitating the biosynthetic maturation and cell surface expression of MLC1, and suggest that pharmacological strategies aimed to increase surface expression of MLC1 and/or VRAC activity may be beneficial for MLC patients.

A functional CFTR assay using primary cystic fibrosis intestinal organoids

We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro-expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.

Putative pore‐loops of TMEM16/anoctamin channels affect channel density in cell membranes

The recently identified TMEM16/anoctamin protein family includes Ca(2+)-activated anion channels (TMEM16A, TMEM16B), a cation channel (TMEM16F) and proteins with unclear function. TMEM16 channels consist of eight putative transmembrane domains (TMs) with TM5-TM6 flanking a re-entrant loop thought to form the pore. In TMEM16A this region has also been suggested to contain residues involved in Ca(2+) binding. The role of the putative pore-loop of TMEM16 channels was investigated using a chimeric approach. Heterologous expression of either TMEM16A or TMEM16B resulted in whole-cell anion currents with very similar conduction properties but distinct kinetics and degrees of sensitivity to Ca(2+). Furthermore, whole-cell currents mediated by TMEM16A channels were ∼six times larger than TMEM16B-mediated currents. Replacement of the putative pore-loop of TMEM16A with that of TMEM16B (TMEM16A-B channels) reduced the currents by ∼six-fold, while the opposite modification (TMEM16B-A channels) produced a ∼six-fold increase in the currents. Unexpectedly, these changes were not secondary to variations in channel gating by Ca(2+) or voltage, nor were they due to changes in single-channel conductance. Instead, they depended on the number of functional channels present on the plasma membrane. Generation of additional, smaller chimeras within the putative pore-loop of TMEM16A and TMEM16B led to the identification of a region containing a non-canonical trafficking motif. Chimeras composed of the putative pore-loop of TMEM16F transplanted into the TMEM16A protein scaffold did not conduct anions or cations. These data suggest that the putative pore-loop does not form a complete, transferable pore domain. Furthermore, our data reveal an unexpected role for the putative pore-loop of TMEM16A and TMEM16B channels in the control of the whole-cell Ca(2+)-activated Cl(-) conductance.

Electrophysiological Characteristics of Enteric Neurons Isolated from the Immortomouse

Recently, two enteric neuronal cell lines, one fetal and the other post-natal (IM-PEN), have been developed from the H-2K(b)-tsA58 transgenic mouse (immortomouse). However, their electrophysiological properties are not known. The goal of this study was to determine the electrical excitability and ionic conductance of the immortalized postnatal enteric neuronal (IM-PEN) cell line. Whole cell patch clamp studies, immunohistochemistry and RT-PCR were performed on differentiated IM-PEN cells following propagation at 33 °C and differentiation at 37 °C. Differentiated IM-PEN cells stained positively for the neuron specific markers βIII-tubulin and PGP9.5. The mRNA for several ion channels expressed in enteric neurons were detected by RT-PCR. In current clamp, the resting membrane potential was -24.6 ± 2.1 mV (n = 6) for IM-FEN and -29.8 ± 0.9 mV (n = 30) for IM-PEN. Current injections from Vh -80 mV resulted in passive responses but not action potentials. Depolarizing pulses in the whole cell voltage clamp configuration from Vh -80 mV elicited small nifedipine-sensitive inward currents. Additionally, outward currents with slow deactivating tail currents were blocked by niflumic acid and low chloride solution. A volume-regulated anion current was elicited by hypo-osmotic solution and inhibited by 10 μM DCPIB. Growth with rabbit gastrointestinal smooth muscle did not yield significant differences in the active properties of the IM-PEN cell line. Transient expression of L-type Ca(2+) channels produced large inward currents demonstrating a working mechanism for protein folding and transport. The electrophysiological characteristics of IM-PEN cells suggest that chloride channels in IM-PEN cells play an important role in their resting state, and membrane trafficking of some of the ion channels may preclude their electrical excitability.

Is ASP-454 Necessary for Na+ and K+ Binding in the Glutamate Transporter EAAC1?

The glutamate transporter Excitatory Amino Acid Carrier 1 (EAAC1) catalyzes rapid removal of glutamate, the major excitatory neurotransmitter in the brain, from the extracellular space, preventing its accumulation to neurotoxic levels. Uphill substrate transport is coupled to co-transport of 3 Na+, 1 H+, and counter-transport of 1 K+ ion. The cation binding sites are yet to be fully identified in this transporter. Here, we investigate the role of the highly conserved D454 residue which is thought to contribute its side chain to coordinating these cations. D454 was mutated to the non-ionizable asparagine, as well as alanine. K+-induced relocation was impaired in EAAC1-D454N, as the transporter no longer catalyzed forward transport. Our findings indicated that K+ could still bind to EAAC1-D454N, but with reduced apparent affinity. Na+/glutamate exchange was functional in EAAC1-D454N, as demonstrated by the presence of transient currents following rapid glutamate application and voltage jumps. In the D454A mutant, glutamate application inhibited inward leak anion currents, and transient currents in response to voltage jumps were abolished. This is consistent with Na+ binding being impaired in this transporter. K+ binding was also eliminated by the D454A mutation. Although D454 is proposed to participate in Na+ binding, its protonation state is yet to be established. pKa calculations for the bacterial homologue Gltph indicate that D405 (analogous to D454) is protonated at physiological pH with a highly perturbed pKa in the range of 7.6 to >14. Valences calculated using the Poison-Boltzmann equation for EAAC1 with D454 protonated (0.45) were close in value to the experimental values obtained for EAAC1-D54N (0.71) and EAAC1-WT (0.64). Together, our results suggest that D454 is protonated under physiological conditions, and participates in Na+ as well as K+ binding.

Gas-phase dissociative electron attachment to flavonoids and possible similarities to their metabolic pathways

The gas-phase empty-level structures and formation of anion states via resonance attachment of low-energy electrons to the flavonoids naringenin (III), quercetin (IV) and myricetin (V) and the smaller reference molecules chromone (I) and flavone (II) are investigated experimentally for the first time. Dissociative electron attachment spectroscopy (DEAS) is used to measure the fragment anion currents generated through dissociative decay channels of the molecular anions of compounds I–V, detected with a mass filter as a function of the incident electron energy in the 0–14 eV energy range. Due to the insufficient volatility of flavonoids III–V, the energies of vertical electron attachment associated with temporary occupation of the lower-lying virtual orbitals are measured with electron transmission spectroscopy (ETS) only in the smaller reference molecules I and II. The experimental findings are interpreted with the support of appropriate density functional theory calculations with the B3LYP functional. The experimental vertical electron attachment energies measured in the ET spectra of I and II are compared with the orbital energies of the neutral molecules scaled using an empirically calibrated linear equation. The vertical and adiabatic electron affinities are evaluated at the B3LYP/6-31+G(d) level as the anion/neutral total energy difference. The latter theoretical method is also used for evaluation of the most stable conformers of the neutral molecules, O–H bond dissociation energies and thermodynamic energy thresholds for production of the anion fragments observed in the DEA spectra. A possible role played by loss of an H(2) molecule from the parent molecular anion in vivo in the mitochondrial respiratory chain is briefly discussed.

Bicuculline‐ and neurosteroid‐sensitive tonic chloride current in rat hypoglossal motoneurons and atypical dual effect of SR95531

Hypoglossal motoneurons (HMs) are known to be under 'permanent' bicuculline-sensitive inhibition and to show 'transient' synaptic γ-aminobutyric acid (GABA)(A) and glycine inhibitory responses. The present paper describes a permanent bicuculline-sensitive current that should contribute to their tonic inhibition. This current was recorded in brainstem slices superfused without any exogenous agonist and remained detectable with tetrodotoxin. It could also be blocked by the other GABA(A) antagonists picrotoxin (PTX) and 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide) (SR95531; gabazine), but persisted in the presence of a specific blocker of α5-containing GABA(A) receptors. Addition of 2 μm 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP), known to preferentially activate GABA(A) receptors devoid of a γ-subunit, induced a sustained anionic current that could be further enhanced by neurosteroids such as allopregnanolone (100 nm). Thus, HMs show a tonic inhibitory current carried by extrasynaptic γ-free GABA(A) receptors, highly sensitive to neurosteroids. A second result was obtained by using SR95531 at concentrations sufficiently high to rapidly block the tonic current above the chloride equilibrium potential (E(C) (l)). Surprisingly, below E(C) (l) , SR95531 (10-40 μm) activated a sustained inward current, associated with a conductance increase, and resistant to bicuculline or PTX (100 μm). Similarly, after blockade of the bicuculline-sensitive current, SR95531 activated an outward current above E(C) (l). The bicuculline-resistant anionic current activated by SR95531 could be blocked by a GABA(C) receptor antagonist. Thus, two types of inhibitory GABA receptors, belonging to the GABA(A) and GABA(C) families, are able to show a sustained activity in HMs and provide promising targets for neuroprotection under overexcitatory situations known to easily damage these particularly fragile neurons.

Time for TMEM?

Time for TMEM?

A point mutation associated with episodic ataxia 6 increases glutamate transporter anion currents

Episodic ataxia is a human genetic disease characterized by paroxysmal cerebellar incoordination. There are several genetically and clinically distinct forms of this disease, and one of them, episodic ataxia type 6, is caused by mutations in the gene encoding a glial glutamate transporter, the excitatory amino acid transporter-1. So far, reduced glutamate uptake by mutant excitatory amino acid transporter-1 has been thought to be the main pathophysiological process in episodic ataxia type 6. However, excitatory amino acid transporter-1 does not only mediate secondary-active glutamate transport, but also functions as an ion channel. Here, we examined the effects of a disease-associated point mutation, P290R, on glutamate transport, anion current as well as on the subcellular distribution of excitatory amino acid transporter-1 using heterologous expression in mammalian cells. P290R reduces the number of excitatory amino acid transporter-1 in the surface membrane and impairs excitatory amino acid transporter-1-mediated glutamate uptake. Cells expressing P290R excitatory amino acid transporter-1 exhibit larger anion currents than wild-type cells in the absence as well as in the presence of external l-glutamate, despite a lower number of mutant transporters in the surface membrane. Noise analysis revealed unaltered unitary current amplitudes, indicating that P290R modifies opening and closing, and not anion permeation through mutant excitatory amino acid transporter-1 anion channels. These findings identify gain-of-function of excitatory amino acid transporter anion conduction as a pathological process in episodic ataxia. Episodic ataxia type 6 represents the first human disease found to be associated with altered function of excitatory amino acid transporter anion channels and illustrates possible physiological and pathophysiological impacts of this functional mode of this class of glutamate transporters.

Ion Channels in Plants

Since the first recordings of single potassium channel activities in the plasma membrane of guard cells more than 25 years ago, patch-clamp studies discovered a variety of ion channels in all cell types and plant species under inspection. Their properties differed in a cell type- and cell membrane-dependent manner. Guard cells, for which the existence of plant potassium channels was initially documented, advanced to a versatile model system for studying plant ion channel structure, function, and physiology. Interestingly, one of the first identified potassium-channel genes encoding the Shaker-type channel KAT1 was shown to be highly expressed in guard cells. KAT1-type channels from Arabidopsis thaliana and its homologs from other species were found to encode the K(+)-selective inward rectifiers that had already been recorded in early patch-clamp studies with guard cells. Within the genome era, additional Arabidopsis Shaker-type channels appeared. All nine members of the Arabidopsis Shaker family are localized at the plasma membrane, where they either operate as inward rectifiers, outward rectifiers, weak voltage-dependent channels, or electrically silent, but modulatory subunits. The vacuole membrane, in contrast, harbors a set of two-pore K(+) channels. Just very recently, two plant anion channel families of the SLAC/SLAH and ALMT/QUAC type were identified. SLAC1/SLAH3 and QUAC1 are expressed in guard cells and mediate Slow- and Rapid-type anion currents, respectively, that are involved in volume and turgor regulation. Anion channels in guard cells and other plant cells are key targets within often complex signaling networks. Here, the present knowledge is reviewed for the plant ion channel biology. Special emphasis is drawn to the molecular mechanisms of channel regulation, in the context of model systems and in the light of evolution.

Glutamate Transporters EAAT4 and EAAT5 Are Expressed in Vestibular Hair Cells and Calyx Endings

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.

Function without form: an ongoing search for maxi-anion channel proteins. Focus on “Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells”

<i>Function without form: an ongoing search for maxi-anion channel proteins</i>. Focus on “Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells”

Melatonin down-regulates volume-sensitive chloride channels in fibroblasts

Melatonin has been reported to present with vasorelaxant and anti-fibrotic properties. We hypothesized that melatonin may down-regulate volume-regulated anion channels (VRAC) in fibroblasts to limit their migration and proliferation. While acute exposure of L929 fibroblasts to melatonin did not result in a significant decrease in VRAC current, pretreatment with 100 μM melatonin for 1 h decreased swelling-dependent activation of anion currents by 83% as measured by whole-cell perforated patch-clamp technique. This down-regulation of VRAC currents was dose-dependent with a half-maximal inhibition of 3.02 ± 0.48 μM. Overnight treatment of cells with 100 nM melatonin had the same inhibitory potency as a 1-h treatment with 100 μM. A similar down-regulatory effect of melatonin on VRAC was observed in primary rat lung fibroblasts. The effect of melatonin was prevented by luzindole and K185 that suggests implication of MT2 receptor. GF109203X, a protein kinase C inhibitor, blocked melatonin's action on VRAC, indicating that MT2 receptor activation results in stimulation of PKC. Consequently, melatonin inhibited regulatory volume decrease following hypotonic swelling of cells. Melatonin also decreased the migration of L929 fibroblasts through the same pathways that blocked VRAC. There was no significant inhibition of cell proliferation. Our study suggests that the attenuation of fibrosis and vascular remodeling by melatonin seen in animal models of hypertension and pulmonary fibrosis might be, in part, related to blunted fibroblast migration possibly through protein kinase C-mediated decrease in chloride channel activity.

Sialin ( SLC17A5 ) functions as a nitrate transporter in the plasma membrane

In vivo recycling of nitrate (NO(3)(-)) and nitrite (NO(2)(-)) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate-nitrite-NO balance. More than 25% of the circulating NO(3)(-) is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO(3)(-) to NO(2)(-), which enters circulation and leads to NO generation. The transporters for NO(3)(-) in salivary glands have not yet been identified. Here we report that sialin (SLC17A5), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO(3)(-)/H(+) cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO(3)(-) or sialic acid (SA), but not by Br(-), and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO(3)(-)-induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H(+)-dependent NO(3)(-) conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO(3)(-) secretion in saliva after intake of a NO(3)(-)-rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO(3)(-)/H(+) transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis.

Disease‐causing mutations C277R and C277Y modify gating of human ClC‐1 chloride channels in myotonia congenita

Myotonia congenita is a genetic condition that is caused by mutations in the muscle chloride channel gene CLCN1 and characterized by delayed muscle relaxation and muscle stiffness. We here investigate the functional consequences of two novel disease-causing missense mutations, C277R and C277Y, using heterologous expression in HEK293T cells and patch clamp recording. Both mutations reduce macroscopic anion currents in transfected cells. Since hClC-1 is a double-barrelled anion channel, this reduction in current amplitude might be caused by altered gating of individual protopores or of joint openings and closing of both protopores. We used non-stationary noise analysis and single channel recordings to separate the mutants' effects on individual and common gating processes. We found that C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates resulting in decreases of absolute open probabilities of homodimeric channels to values below 3%. In heterodimeric channels, C277R and C277Y also reduce open probabilities and shift the common gate activation curve towards positive potentials. Moreover, C277Y modifies pore properties of hClC-1. It reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence to I(-) = NO(3)(-) >Br(-)>Cl(-). Our findings predict a dramatic reduction of the muscle fibre resting chloride conductance and thus fully explain the disease-causing effects of mutations C277R and C277Y. Moreover, they provide additional insights into the function of C277, a residue recently implicated in common gating of ClC channels.

Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action

The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca(2+)-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.

Neutralizing Aspartate 83 Modifies Substrate Translocation of Excitatory Amino Acid Transporter 3 (EAAT3) Glutamate Transporters

Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission by removing glutamate from the synaptic cleft into neuronal and glial cells. EAATs are not only secondary active glutamate transporters but also function as anion channels. Gating of EAAT anion channels is tightly coupled to transitions within the glutamate uptake cycle, resulting in Na(+)- and glutamate-dependent anion currents. A point mutation neutralizing a conserved aspartic acid within the intracellular loop close to the end of transmembrane domain 2 was recently shown to modify the substrate dependence of EAAT anion currents. To distinguish whether this mutation affects transitions within the uptake cycle or directly modifies the opening/closing of the anion channel, we used voltage clamp fluorometry. Using three different sites for fluorophore attachment, V120C, M205C, and A430C, we observed time-, voltage-, and substrate-dependent alterations of EAAT3 fluorescence intensities. The voltage and substrate dependence of fluorescence intensities can be described by a 15-state model of the transport cycle in which several states are connected to branching anion channel states. D83A-mediated changes of fluorescence intensities, anion currents, and secondary active transport can be explained by exclusive modifications of substrate translocation rates. In contrast, sole modification of anion channel opening and closing is insufficient to account for all experimental data. We conclude that D83A has direct effects on the glutamate transport cycle and that these effects result in changed anion channel function.