K+-sensitive gating of the K+outward rectifier in stomatal guard cells

K⁺-sensitive gating of the K⁺outward rectifier in Vicia guard cells

by Blatt M. R., Gradmann D. (1997)

1. Laboratory of Plant Physiology and Biophysics, University of London, Wye College, Wye, Kent TN25 5AH UKGB
2. Pflanzenphysiologisches Institut, Universität Göttingen, Untere Karspüle 2, D-37073 GöttingenDE

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in J Membr Biol 158: 241–256 –  https://doi.org/10.1007/s002329900261 – 

https://link.springer.com/article/10.1007%2Fs002329900261#citeas 

Abstract

The effect of extracellular cation concentration and membrane voltage on the current carried by outward-rectifying K⁺ channels was examined in stomatal guard cells of Vicia faba L.

Intact guard cells were impaled with double-barrelled microelectrodes and the K⁺ current was monitored under voltage clamp in 0.1–30 mm K⁺ and in equivalent concentrations of Rb⁺, Cs⁺and Na⁺. From a conditioning voltage of −200 mV, clamp steps to voltages between −150 and +50 mV in 0.1 mm K⁺ activated current through outward-rectifying K⁺ channels (I _K, out) at the plasma membrane in a voltage-dependent fashion. Increasing [K⁺] _o shifted the voltage-sensitivity of I _K, out in parallel with the equilibrium potential for K⁺ across the membrane.

A similar effect of [K⁺] _o was evident in the kinetics of I _K, out activation and deactivation, as well as the steady-state conductance- (g _K ⁻) voltage relations. Linear conductances, determined as a function of the conditioning voltage from instantaneous I-V curves, yielded voltages for half-maximal conductance near −130 mV in 0.1 mm K⁺, −80 mV in 1.0 mm K⁺, and −20 mV in 10 mm K⁺.

Similar data were obtained with Rb⁺ and Cs⁺, but not with Na⁺, consistent with the relative efficacy of cation binding under equilibrium conditions (K⁺≥ Rb⁺ > Cs⁺ > > Na⁺). Changing Ca²⁺ or Mg²⁺ concentrations outside between 0.1 and 10 mm was without effect on the voltage-dependence of g _K or on I _K, out activation kinetics, although 10 mm [Ca²⁺] _o accelerated current deactivation at voltages negative of −75 mV.

At any one voltage, increasing [K⁺] _o suppressed g _K completely, an action that showed significant cooperativity with a Hill coefficient of 2. The apparent affinity for K⁺ was sensitive to voltage, varying from 0.5 to 20 mm with clamp voltages near −100 to 0 mV, respectively.

These, and additional data indicate that extracellular K⁺ acts as a ligand and alters the voltage-dependence of I _K, out gating; the results implicate K⁺-binding sites accessible from the external surface of the membrane, deep within the electrical field, but distinct from the channel pore; and they are consistent with a serial 4-state reaction-kinetic model for channel gating in which binding of two K⁺ ions outside affects the distribution between closed states of the channel.

The OnGuard model providing a framework for systems analysis of stomatal guard cells

 

 

Systems dynamic modeling of the stomatal guard cell predicts emergent behaviors in transport, signaling, and volume control.

by Chen Z. H., Hills A., Bätz U., Amtmann A., Lew V. L., Blatt M. R. (2012)

in Plant Physiol 159: 1235–1251 – doi:10.1104/pp.112.197350 –

CrossRef | CAS –

http://researchdirect.westernsydney.edu.au/islandora/object/uws:13280

Abstract

The dynamics of stomatal movements and their consequences for photosynthesis and transpirational water loss have long been incorporated into mathematical models, but none have been developed from the bottom up that are widely applicable in predicting stomatal behavior at a cellular level.

We previously established a systems dynamic model incorporating explicitly the wealth of biophysical and kinetic knowledge available for guard cell transport, signaling, and homeostasis.

Here we describe the behavior of the model in response to experimentally documented changes in primary pump activities and malate (Mal) synthesis imposed over a diurnal cycle.

We show that the model successfully recapitulates the cyclic variations in H+, K+, Cl-, and Mal concentrations in the cytosol and vacuole known for guard cells. It also yields a number of unexpected and counterintuitive outputs. Among these, we report a diurnal elevation in cytosolic-free Ca2+ concentration and an exchange of vacuolar Cl- with Mal, both of which find substantiation in the literature but had previously been suggested to require additional and complex levels of regulation.

These findings highlight the true predictive power of the OnGuard model in providing a framework for systems analysis of stomatal guard cells, and they demonstrate the utility of the OnGuard software and HoTSig library in exploring fundamental problems in cellular physiology and homeostasis.

A remarkably complex network, layering positive and negative controls with the ion channels that facilitate ion fluxes for stomatal movement

 

Signalling gates in abscisic acid-mediated control of guard cell ion channels

by Blatt M. R., Grabov A.  (1997)

Michael R. Blatt, Univ. of London, Wye, Kent 

Alexander Grabov, Univ. of London, Wye, Kent

in Physiol. Plantarum Vol. 100, Issue 3 July 1997 , 481–490 – DOI: 10.1111/j.1399-3054.1997.tb03052.x – 

http://onlinelibrary.wiley.com/doi/10.1111/j.1399-3054.1997.tb03052.x/full

Abstract

Multiple signalling pathways and their messengers – entailing changes in cytosolic-free Ca²⁺([Ca²⁻]). pH (pH) and protein phosphorylation – underpin K⁺and anion channel control during stomatal movements. This redundancy is wholly consistent with the ability of the guard cells to integrate the wide range of environmental and hormonal stimuli that affect stomatal aperture.

Signal redundancy effects a spectrum of graded responses by linking pathways to gate signal transmission, and so boosts or mutes the final ‘integrated signal’ that reaches each ion channel.

All evidence supports a role for the AB11 protein phosphatase and protein kinase elements in gating K⁺channel sensitivity to pH and ABA. Changes in [Ca²⁺]I. in turn, are demonstrably sensitive to pH₁. Because each of these signal elements modulate and, in turn, are influenced by the activity of different sets of ion channels, the additional couplings engender a remarkably complex network, layering positive and negative controls with the ion channels that facilitate ion fluxes for stomatal movement.

The rapidity of stomatal responses

 

Stomatal size, speed, and responsiveness impact on photosynthesis and water use efficiency.

by Lawson T., Blatt M. R. (2014)

Tracy Lawson,

Michael R. Blatt

in Plant Physiol. 2014 Apr;164(4):1556-1570. doi: 10.1104/pp.114.237107. Epub 2014 Feb 27.-

CAS, PubMed, Article – PubMed Abstract | CrossRef Full Text | Google Scholar

http://www.ncbi.nlm.nih.gov/pubmed?Db=pubmed&Cmd=ShowDetailView&TermToSearch=24578506

Abstract

The control of gaseous exchange between the leaf and bulk atmosphere by stomata governs CO₂ uptake for photosynthesis and transpiration, determining plant productivity and water use efficiency. The balance between these two processes depends on stomatal responses to environmental and internal cues and the synchrony of stomatal behavior relative to mesophyll demands for CO₂.

Here we examine the rapidity of stomatal responses with attention to their relationship to photosynthetic CO₂ uptake and the consequences for water use. We discuss the influence of anatomical characteristics on the velocity of changes in stomatal conductance and explore the potential for manipulating the physical as well as physiological characteristics of stomatal guard cells in order to accelerate stomatal movements in synchrony with mesophyll CO₂ demand and to improve water use efficiency without substantial cost to photosynthetic carbon fixation.

We conclude that manipulating guard cell transport and metabolism is just as, if not more likely to yield useful benefits as manipulations of their physical and anatomical characteristics. Achieving these benefits should be greatly facilitated by quantitative systems analysis that connects directly the molecular properties of the guard cells to their function in the field.

Protein phosphorylation, Ca2+ channel, ABA and stomata

 

Protein phosphorylation activates the guard cell Ca²⁺ channel and is a prerequisite for gating by abscisic acid.

by Köhler B., Blatt M. R. (2002)

Barbara Köhler, Michael R. Blatt

in Plant J. 32,185–194. – DOI: 10.1046/j.1365-313X.2002.01414.x – 

Wiley Online Library | PubMed

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313X.2002.01414.x/full

Summary

Protein phosphorylation and cytosolic-free [Ca²⁺] ([Ca²⁺]_i) contribute to signalling cascades evoked by the water-stress hormone abscisic acid (ABA) that lead to stomatal closure in higher-plant leaves. ABA activates an inward-rectifying Ca²⁺ channel at the plasma membrane of stomatal guard cells, promoting Ca²⁺ entry by shifting the voltage-sensitivity of the channels.

Because many of these effects could be mediated by kinase/phosphatase action at the membrane, we examined a role for protein (de-)phosphorylation in plasma membrane patches from Vicia guard cells. Ca²⁺ channel activity decayed rapidly in excised patches, and recovered on adding ATP (K_1/2, 1.3 ± 0.7 mm) but not the non-hydrolyzable analog ATPγS.

ABA activation of the channel required the presence of ATP and like ABA, the 1/2 A-type protein phosphatase antagonists okadaic acid (OA) and calyculin A (CA) enhanced Ca²⁺ channel activity by increasing the open probability and number of active channels.

Neither ATP nor the antagonists affected the mean open lifetime of the channel, suggesting an action through changes in closed lifetime distributions. Like ABA, OA and CA shifted the voltage-sensitivities of the Ca²⁺ current and [Ca²⁺]_i increases in intact guard cells towards positive voltages. OA and CA also augmented the [Ca²⁺]_i rise evoked by hyperpolarization and delayed its recovery.

These results demonstrate a membrane-delimited interaction between 1/2 A-type protein phosphatase(s) and the Ca²⁺ channel or associated proteins, and they are consistent with a role for protein (de-)phosphorylation in ABA signalling mediated directly through Ca²⁺ channel gating that leads to [Ca²⁺]_i increases in the guard cells.

 

Extracellular Ba2‡ and voltage interact in stomatal guard cells

 

Extracellular Ba2‡ and voltage interact to gate Ca2‡ channels at the plasma membrane of stomatal guard cells

by Hamilton D. W. A.,  Hills A., Blatt M. R. (2001)

in FEBS Letters 491 (2001) 99-103 – 

http://onlinelibrary.wiley.com/store/10.1016/S0014-5793(01)02176-7/asset/feb2s0014579301021767.pdf?v=1&t=ip5xzwp2&s=16a1dd5e6e60dc5a7e36dea9e88360ec36821ea3

Abstract

Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] ([Ca2+]i) that regulate K+ and Cl3 channels for stomatal closure in higher-plant leaves.

Under voltage clamp, the initial rate of increase in [Ca2+]i in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating.

To test this idea, we recorded single-channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages.

We found that the open probability (Po) increased strongly with external Ba2+ concentration, consistent with a 4-fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state.

Dwell time analyses indicated the presence of a single open state and at least three closed states of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state.

Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage.

We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate.

ABA, Ca2+ and stomatal guard cells

Photo credit: NCBI

P_o is suppressed by micromolar [Ca²⁺]_i. Means ± SE of P_o from mean open times of 100-s recordings at −120 mV (n = 3). Ca²⁺ added on the cytosolic side (inside) during inside-out recordings against a background of 30 mM Ba²⁺ and with 10 mM Ba²⁺ outside. (Insets) Segments of traces at each [Ca²⁺]_i. Data from one patch. Scale: vertical, 1 pA; horizontal, 1 s.

Ca²⁺ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid.

by Hamilton D. W. A.,  Hills A., Kohler B., Blatt M. R. (2000)

in Proc. Natl Acad. Sci. USA, 97, 4967–4972. –

CrossRef | PubMed | CAS | ADS

http://www.ncbi.nlm.nih.gov/pubmed/10781106

Abstract

In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by means of Ca(2+) entry from outside and release from intracellular stores. The mechanism(s) for Ca(2+) flux across the plasma membrane is poorly understood.

Because [Ca(2+)](i) increases are voltage-sensitive, we suspected a Ca(2+) channel at the guard cell plasma membrane that activates on hyperpolarization and is regulated by ABA.

We recorded single-channel currents across the Vicia guard cell plasma membrane using Ba(2+) as a charge-carrying ion. Both cell-attached and excised-patch measurements uncovered single-channel events with a maximum conductance of 12.8 +/- 0.4 pS and a high selectivity for Ba(2+) (and Ca(2+)) over K(+) and Cl(-).

Unlike other Ca(2+) channels characterized to date, these channels rectified strongly toward negative voltages with an open probability (P(o)) that increased with [Ba(2+)] outside and decreased roughly 10-fold when [Ca(2+)](i) was raised from 200 nM to 2 microM. Adding 20 microM ABA increased P(o), initially by 63- to 260-fold; in both cell-attached and excised patches, it shifted the voltage sensitivity for channel activation, and evoked damped oscillations in P(o) with periods near 50 s. A similar, but delayed response was observed in 0.1 microM ABA.

These results identify a Ca(2+)-selective channel that can account for Ca(2+) influx and increases in [Ca(2+)](i) triggered by voltage and ABA, and they imply a close physical coupling at the plasma membrane between ABA perception and Ca(2+) channel control.

ABA and anion channel kinetics in stomata

 

Alteration of anion channel kinetics in wild-type and abi1-1 transgenic Nicotiana benthamiana guard cells by abscisic acid.

by Grabov A., Leung J., Giraudat J.,  Blatt M. R.(1997)

in Plant J.12, 203–213. –

Wiley Online Library | PubMed | CAS | CrossRefMedlineWeb of Science 

http://www.ncbi.nlm.nih.gov/pubmed/9263461

Abstract

The influence of the plant water-stress hormone abscisic acid (ABA) on anion channel activity and its interaction with protein kinase and phosphatase antagonists was examined in stomatal guard cells of wild-type Nicotiana benthamiana L. and of transgenic plants expressing the dominant-negative (mutant) Arabidopsis abi1-1 protein phosphatase.

Intact guard cells were impaled with double-barrelled micro-electrodes and membrane current was recorded under voltage clamp in the presence of 15 mM CsCl and 15 mM tetraethylammonium chloride (TEA-Cl) to eliminate K+ channel currents. Under these conditions, the free-running voltage was situated close to 0 mV (+9 +/- 6 mV, n = 18) and the membrane under voltage clamp was dominated by anion channel current (ICl) as indicated from tall current reversal near the expected chloride equilibrium potential, current sensitivity to the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, and by its voltage-dependent kinetics.

Pronounced activation of ICl was recorded on stepping from a conditioning voltage of -250 mV to voltages between -30 and +50 mV, and the current deactivated with a voltage-dependent halftime at more negative voltages (tau approximately equal to 0.3 sec at -150 mV). Challenge with 20 microM ABA increased the steady-state current conductance, gCl, near 0 mV by 1.2- to 2.6-fold and at -150 mV by 4.5- to sixfold with a time constant of 40 +/- 4 sec, and it slowed ICl deactivation as much as fourfold at voltages near -50 mV, introducing two additional voltage-sensitive kinetic components to these current relaxations.

Neither the steady-state and kinetic characteristics of ICl nor its sensitivity to ABA were influenced by H7 or staurosporine, both broad-range protein kinase antagonists. However, the protein phosphatase 1/2A antagonist calyculin A mimicked the effects of ABA on gCl and current relaxations on its own and exhibited a synergistic interaction with ABA, enhancing ICl sensitivity to ABA three- to four-fold.

Quantitatively similar current characteristics were recorded from guard cells of abi1-1 transgenic N. benthamiana, indicating that the abi1-1 protein phosphatase does not influence the anion current or its response to ABA directly.

These results demonstrate that ABA stimulates ICl and modulates its voltage sensitivity. Furthermore, they show that ABA promotes ICl, either by introducing additional long-lived states of the channel or by activating a second anion channel with similar permeation characteristics but with a very long dwell time in the open state.

Overall, the data are broadly consistent with the view that ABA action engenders coordinate control of ICl together with guard cell K+ channels to effect solute loss and stomatal closure.

Hyperpolarization in stomata

 

A steep dependence of inward-rectifying potassium channels on cytosolic free calcium concentration increase evoked by hyperpolarization in guard cells

by Grabov A., Blatt M.R. (1999)

in Plant Physiol. 119:277–287. – doi: http://dx.doi.org/10.1104/pp.119.1.277

Abstract/FREE Full Text

http://www.plantphysiol.org/content/119/1/277.abstract?ijkey=5237c9fa14aadf4e2fc4c176ae894cc177161b50&keytype2=tf_ipsecsha

Abstract

Inactivation of inward-rectifying K⁺ channels (I _K,in) by a rise in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca²⁺]_i action onI _K,in has never been quantified, nor are its origins well understood.

We used membrane voltage to manipulate [Ca²⁺]_i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recordingI _K,in under a voltage clamp and [Ca²⁺]_i by Fura-2 fluorescence ratiophotometry. I _K,in inactivation correlated positively with [Ca²⁺]_i and indicated a K _i of 329 ± 31 nM with cooperative binding of four Ca²⁺ ions per channel.

I _K,in was promoted by the Ca²⁺ channel antagonists Gd³⁺ and calcicludine, both of which suppressed the [Ca²⁺]_i rise, but the [Ca²⁺]_i rise was unaffected by the K⁺channel blocker Cs⁺.

We also found that ryanodine, an antagonist of intracellular Ca²⁺channels that mediate Ca²⁺-induced Ca²⁺ release, blocked the [Ca²⁺]_i rise, and Mn²⁺quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores.

These and additional results point to a high signal gain in [Ca²⁺]_i control ofI _K,in and to roles for discrete Ca²⁺ flux pathways in feedback control of the K⁺ channels by membrane voltage.

 

Ca++, membrane voltage, ABA and stomata

 

Membrane voltage initiates Ca²⁺ waves and potentiates Ca²⁺ increases with abscisic acid in stomatal guard cells.

by Grabov A., Blatt M. R. (1998a)

in Proc. Natl Acad. Sci. USA, 95, 4778–4783. –

CrossRef |PubMed | CAS | ADS 

http://www.pnas.org/content/95/8/4778

Abstract

In higher plants changes and oscillations in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca²⁺]_i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca²⁺]_i to voltage and its interaction with ABA.

We recorded [Ca²⁺]_iby Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near −50 mV and −200 mV were associated with oscillations in [Ca²⁺]_i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca²⁺]_i to increase, often in excess of 1 μM, from resting values near 100 nM.

Image analysis showed that the voltage stimulus evoked a wave of high [Ca²⁺]_i that spread centripetally from the peripheral cytoplasm within 5–10 s and relaxed over 40–60 s thereafter. The [Ca²⁺]_i increases showed a voltage threshold near −120 mV and were sensitive to external Ca²⁺concentration.

Substituting Mn²⁺ for Ca²⁺ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca²⁺]_i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca²⁺]_i.

These results demonstrate a capacity for voltage to evoke [Ca²⁺]_i increases, they point to a dual interaction with ABA in triggering and propagating [Ca²⁺]_i increases, and they implicate a role for voltage in “conditioning” [Ca²⁺]_isignals that regulate ion channels for stomatal function.