Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells.

Abstract Cytosolic calcium ([Ca2+]cyt) elevation activates plasma membrane anion channels in guard cells, which is required for stomatal closure. However, involvement of the anion channels in the [Ca2+]cyt elevation remains unclear. We investigated the involvement using Arabidopsis thaliana anion channel mutants, slac1-4 slah3-3 and slac1-4 almt12-1. Extracellular calcium induced stomatal closure in the wild-type plants but not in the anion channel mutant plants whereas extracellular calcium induced [Ca2+]cyt elevation both in the wild-type guard cells and in the mutant guard cells. The peak height and the number of the [Ca2+]cyt spike were lower and larger in the slac1-4 slah3-3 than in the wild-type and the height and the number in the slac1-4 almt12-1 were much lower and much larger than in the wild-type. These results suggest that the anion channels are involved in the regulation of [Ca2+]cyt elevation in guard cells.

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Molecular basis for the R-type anion channel QUAC1 activity in guard cells

10.1101/2021.09.09.459598 ◽

2021 ◽

Author(s):

Li Qin ◽

Ling-hui Tang ◽

Jia-shu Xu ◽

Xian-hui Zhang ◽

Yun Zhu ◽

...

Keyword(s):

Molecular Basis ◽

Stomatal Closure ◽

Anion Channel ◽

Guard Cells ◽

Channel Activity ◽

Cytoplasmic Domains ◽

Anion Channels ◽

Channel Activation ◽

Environmental Stimuli ◽

Functional Analyses

SUMMARYThe rapid (R)-type anion channel plays a central role in controlling stomatal closure in plant guard cells, thus regulating the exchange of water and photosynthetic gas (CO2) in response to environmental stimuli. The activity of the R- type anion channel is regulated by malate. However, the molecular basis of the R-type anion channel activity remains elusive. Here, we describe the first cryo-EM structure of the R-type anion channel QUAC1 at 3.5 Å resolution in the presence of malate. The structure reveals that the QUAC1 is a symmetrical dimer, forming a single electropositive T-shaped pore for passing anions across the membrane. The transmembrane and cytoplasmic domains are assembled into a twisted bi-layer architecture, with the associated dimeric interfaces nearly perpendicular. Our structural and functional analyses reveal that QUAC1 functions as an inward rectifying anion channel and suggests a mechanism for malate-mediated channel activation. Altogether, our study uncovers the molecular basis for a novel class of anion channels and provides insights into the gating and modulation of the R-type anion channel.

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Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.1.c214 ◽

1997 ◽

Vol 273 (1) ◽

pp. C214-C222 ◽

Cited By ~ 46

Author(s):

V. G. Manolopoulos ◽

T. Voets ◽

P. E. Declercq ◽

G. Droogmans ◽

B. Nilius

Keyword(s):

Endothelial Cells ◽

Anion Channel ◽

Disulfonic Acid ◽

Channel Blockers ◽

Organic Osmolytes ◽

Dependent Manner ◽

Anion Channels ◽

Whole Cell Patch Clamp ◽

Osmolyte Efflux ◽

Dose Dependent

We used a combined biochemical, pharmacological, and electrophysiological approach to study the effects of hyposmotic swelling on organic osmolyte efflux in endothelial cells (EC). In [3H]taurine-loaded monolayers of calf pulmonary artery EC (CPAEC), hyposmolality activated time- and dose-dependent effluxes of [3H]taurine. Swelling-activated [3H]taurine efflux (Jtau swell)in CPAEC was inhibited by the anion channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), fenamates, and also quinine (in a pH-dependent manner), ATP, and the phospholipase A2 inhibitor 4-bromophenacyl bromide. In contrast, Jtau swell was partly or totally insensitive to bumetanide, forskolin, phorbol 12-myristate 13-acetate, and staurosporine. Swelling also activated myo-[3H]inositol efflux that was blocked by tamoxifen, NPPB, DIDS, and niflumic acid. Moreover, the cellular content of taurine and other amino acids was significantly reduced in osmotically activated CPAEC. Finally, in whole cell patch-clamp experiments, taurine, glycine, aspartate, and glutamate exhibited significant permeability for swelling-activated anion channels. In conclusion, hyposmotic swelling activates efflux of taurine and other organic osmolytes in EC. In addition, our results suggest that anion channels may provide a pathway for swelling-activated efflux of organic osmolytes in EC.

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Plasma membrane ion channel regulation during abscisic acid-induced closing of stomata

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1992.0131 ◽

1992 ◽

Vol 338 (1283) ◽

pp. 83-89 ◽

Cited By ~ 15

Keyword(s):

Plasma Membrane ◽

Abscisic Acid ◽

Ion Channels ◽

Patch Clamp ◽

Integrated Control ◽

Anion Channel ◽

Guard Cells ◽

Anion Channels ◽

Higher Plant ◽

Ion Channel Regulation

The plant growth regulator abscisic acid triggers closing of stomata in the leaf epidermis in response to water stress. Recent tracer flux studies, patch-clamp studies, fluorometric Ca 2+ measurements and microelectrode experiments have provided insight into primary transduction mechanisms by which abscisic acid causes stomatal closing. Data show that abscisic acid activates non-selective Ca 2+ permeable ion channels in the plasma membrane of guard cells. The resulting elevation in the free Ca 2+ concentration in the cytosol of guard cells, and the resulting membrane depolarization as well as other unidentified Ca 2+ independent mechanisms are suggested to contribute to activation of voltage- and second messenger-dependent anion channels and outward rectifying K + channels. Recent data suggest the involvement of two types of anion channels in the regulation of stomatal movements, which provide highly distinct mechanisms for anion efflux and depolarization. A novely characterized ‘S-type’ anion channel is likely to provide a key mechanism for long-term depolarization and sustained anion efflux during closing of stomata. Patch-clamp studies have revealed the presence of a network of K + , anion and non-selective Ca 2+ -permeable channels in the plasma membrane of a higher plant cell. The integrated control of these guard cell ion channels by abscisic acid can provide control over K + and anion efflux required for stomatal closing.

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Optogenetic control of the guard cell membrane potential and stomatal movement by the light-gated anion channel GtACR1

Science Advances ◽

10.1126/sciadv.abg4619 ◽

2021 ◽

Vol 7 (28) ◽

pp. eabg4619

Author(s):

Shouguang Huang ◽

Meiqi Ding ◽

M. Rob G. Roelfsema ◽

Ingo Dreyer ◽

Sönke Scherzer ◽

...

Keyword(s):

Guard Cell ◽

Stomatal Closure ◽

Green Light ◽

Anion Channel ◽

Guard Cells ◽

Wild Type ◽

Anion Channels ◽

Light Pulses ◽

Cell Turgor ◽

Open Question

Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO2 and water. In turn, guard cell anion channels are seen as key players for stomatal closure, but is activation of these channels sufficient to limit plant water loss? To answer this open question, we used an optogenetic approach based on the light-gated anion channelrhodopsin 1 (GtACR1). In tobacco guard cells that express GtACR1, blue- and green-light pulses elicit Cl− and NO3− currents of −1 to −2 nA. The anion currents depolarize the plasma membrane by 60 to 80 mV, which causes opening of voltage-gated K+ channels and the extrusion of K+. As a result, continuous stimulation with green light leads to loss of guard cell turgor and closure of stomata at conditions that provoke stomatal opening in wild type. GtACR1 optogenetics thus provides unequivocal evidence that opening of anion channels is sufficient to close stomata.

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Identification of SLAC1 anion channel residues required for CO2/bicarbonate sensing and regulation of stomatal movements

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1807624115 ◽

2018 ◽

Vol 115 (44) ◽

pp. 11129-11137 ◽

Cited By ~ 18

Author(s):

Jingbo Zhang ◽

Nuo Wang ◽

Yinglong Miao ◽

Felix Hauser ◽

J. Andrew McCammon ◽

...

Keyword(s):

Stomatal Closure ◽

Anion Channel ◽

Guard Cells ◽

Channel Activity ◽

Plant Leaves ◽

In Planta ◽

Anion Channels ◽

Accelerated Molecular Dynamics ◽

Bicarbonate Ions ◽

Stomatal Movements

Increases in CO2 concentration in plant leaves due to respiration in the dark and the continuing atmospheric [CO2] rise cause closing of stomatal pores, thus affecting plant–water relations globally. However, the underlying CO2/bicarbonate (CO2/HCO3−) sensing mechanisms remain unknown. [CO2] elevation in leaves triggers stomatal closure by anion efflux mediated via the SLAC1 anion channel localized in the plasma membrane of guard cells. Previous reconstitution analysis has suggested that intracellular bicarbonate ions might directly up-regulate SLAC1 channel activity. However, whether such a CO2/HCO3− regulation of SLAC1 is relevant for CO2 control of stomatal movements in planta remains unknown. Here, we computationally probe for candidate bicarbonate-interacting sites within the SLAC1 anion channel via long-timescale Gaussian accelerated molecular dynamics (GaMD) simulations. Mutations of two putative bicarbonate-interacting residues, R256 and R321, impaired the enhancement of the SLAC1 anion channel activity by CO2/HCO3− in Xenopus oocytes. Mutations of the neighboring charged amino acid K255 and residue R432 and the predicted gate residue F450 did not affect HCO3− regulation of SLAC1. Notably, gas-exchange experiments with slac1-transformed plants expressing mutated SLAC1 proteins revealed that the SLAC1 residue R256 is required for CO2 regulation of stomatal movements in planta, but not for abscisic acid (ABA)-induced stomatal closing. Patch clamp analyses of guard cells show that activation of S-type anion channels by CO2/HCO3−, but not by ABA, was impaired, indicating the relevance of R256 for CO2 signal transduction. Together, these analyses suggest that the SLAC1 anion channel is one of the physiologically relevant CO2/HCO3− sensors in guard cells.

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Anion channels influence ECC by modulating L-type Ca2+ channel in ventricular myocytes

Journal of Applied Physiology ◽

10.1152/japplphysiol.00220.2002 ◽

2002 ◽

Vol 93 (5) ◽

pp. 1660-1668 ◽

Cited By ~ 16

Author(s):

Shi-Sheng Zhou ◽

Zhan Gao ◽

Ling Dong ◽

Yan-Feng Ding ◽

Xiao-Dong Zhang ◽

...

Keyword(s):

Ventricular Myocytes ◽

Anion Channel ◽

Niflumic Acid ◽

Cardiac Contraction ◽

Channel Blockers ◽

Dependent Manner ◽

Anion Channels ◽

Cell Shortening ◽

Fluorescence Measurements ◽

Intracellular Ca

Anion channels are extensively expressed in the heart, but their roles in cardiac excitation-contraction coupling (ECC) are poorly understood. We, therefore, investigated the effects of anion channels on cardiac ventricular ECC. Edge detection, fura 2 fluorescence measurements, and whole cell patch-clamp techniques were used to measure cell shortening, the intracellular Ca2+ transient, and the L-type Ca2+ current ( I Ca,L) in single rat ventricular myocytes. The anion channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid reversibly inhibited the Ca2+ transients and cell shortening in a dose-dependent manner. Comparable results were observed when the majority of the extracellular Cl− was replaced with the relatively impermeant anions glutamate (Glt−) and aspartate (Asp−). NPPB and niflumic acid or the Cl− substitutes did not affect the resting intracellular Ca2+ concentration but significantly inhibited I Ca,L. In contrast, replacement of extracellular Cl− with the permeant anions NO[Formula: see text], SCN−, and Br− supported the ECC and I Ca,L, which were still sensitive to blockade by NPPB. Exposure of cardiac ventricular myocytes to a hypotonic bath solution enhanced the amplitude of cell shortening and supported I Ca,L, whereas hypertonic stress depressed the contraction and I Ca,L. Moreover, cardiac contraction was completely abolished by NPPB (50 μM) under hypotonic conditions. It is concluded that a swelling-activated anion channel may be involved in the regulation of cardiac ECC through modulating L-type Ca2+ channel activity.

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Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells

AJP Cell Physiology ◽

10.1152/ajpcell.00459.2011 ◽

2012 ◽

Vol 303 (9) ◽

pp. C924-C935 ◽

Cited By ~ 28

Author(s):

Md. Rafiqul Islam ◽

Hiromi Uramoto ◽

Toshiaki Okada ◽

Ravshan Z. Sabirov ◽

Yasunobu Okada

Keyword(s):

Connexin 43 ◽

Regulatory Volume Decrease ◽

Atp Release ◽

Anion Channel ◽

Molecular Entity ◽

Channel Blockers ◽

Channel Activity ◽

Anion Channels ◽

Pannexin 1 ◽

Fibrosarcoma Cells

The maxi-anion channel plays a classically recognized role in controlling the membrane potential through the chloride conductance. It also has novel functions as a regulated pathway for the release of the anionic signaling molecules ATP and excitatory amino acids from cells subjected to osmotic perturbation, ischemia, or hypoxia. Because hemichannels formed by pannexins and connexins have been reported to mediate ATP release from a number of cell types, these hemichannels may represent the molecular correlate of the maxi-anion channel. Here, we found that L929 fibrosarcoma cells express functional maxi-anion channels which mediate a major portion of swelling-induced ATP release, and that ATP released via maxi-anion channels facilitates the regulatory volume decrease after osmotic swelling. Also, it was found that the cells express the mRNA for pannexin 1, pannexin 2, and connexin 43. Hypotonicity-induced ATP release was partially suppressed not only by known blockers of the maxi-anion channel but also by several blockers of pannexins including the pannexin 1-specific blocking peptide 10Panx1 and small interfering (si)RNA against pannexin 1 but not pannexin 2. The inhibitory effects of maxi-anion channel blockers and pannexin 1 antagonists were additive. In contrast, maxi-anion channel activity was not affected by pannexin 1 antagonists and siRNAs against pannexins 1 and 2. Although a connexin 43-specific blocking peptide, Gap27, slightly suppressed hypotonicity-induced ATP release, maxi-anion channel activity was not affected by Gap27 or connexin 43-specific siRNA. Thus, it is concluded that the maxi-anion channel is a molecular entity distinct from pannexin 1, pannexin 2, and connexin 43, and that the maxi-anion channel and the hemichannels constitute separate pathways for swelling-induced ATP release in L929 cells.

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