Anions modify the response of guard-cell anion channels to auxin

The anion channel in the guard-cell plasma membrane of Vicia faba, GCAC1, possesses recognition sites for the plant growth hormone auxin at the extracellular mouth of the channel (Marten et al. 1991, Nature 353:759-762). Using the patch-clamp technique we could demonstrate that auxins induced a shift of the voltage dependence of the anion channel to hyperpolarized potentials; the shift was attenuated during an increase in the extracellular chloride concentration, indicating that chloride shields the hormone-binding site. The auxin-induced shift was concentration-dependent, characterized by a Michaelis-Menten type of behaviour with a half saturation constant (K _m) of about 10 M naphthalene-1-acetic acid (1-NAA) in the presence of 2 mM Cl^– and 12 M in 80 mM Cl^–. In the presence of malate, another gating modulator of GCAC1, auxins were less effective, indicating that both ligands compete for common sites. Inactive auxins with respect to stomatal opening or stimulation of the plasma membrane H⁺-ATPase, such as 2-NAA, modulated the activation threshold and kinetics of GCAC1 similar to the active form 1-NAA. At a concentration of 100 M 2-NAA the peak-current potential shifted by about 30 mV more negative.Abbreviations GCAC1 guard cell anion channel 1 - 1-NAA naphthalene-1-acetic acid - 2-NAA naphthalene-2-acetic acid - TEA tetraethylammonium     

钾通道在大鼠支气管平滑肌张力调控中作用的研究

目的：探讨延迟整流钾通道（Kv),高电导钙激活钾通道（BKCa)和ATP敏感钾通道（KATP)在大鼠支气管平滑肌张力调控中的作用。方法：以特异性钾通道阻断剂为工具,采用体外等长张力测定观察钾通道对静息和收缩状态下支气管张力的影响。结果：（1）KV阻断剂4－aminopyridine(4-AP)诱发大鼠支气管平滑肌产生浓度依赖性收缩反应,而BKCa阻断剂tetraethylammonium(TEA)和KATP阻断剂glibenclamide(Glib)对其无影响。（2）去除上皮对4－AP诱发大鼠支气管平滑肌收缩反应无影响,而钙通道阻断剂nifedipine对其有显著抑制效应。（3）在0.1mmol/L组胺或50mmol/L KCl诱发支气管平滑肌收缩之前或之后,加入TEA（1,5mmol/L)或0.1mmol/L 4-AP均显著增强二者诱发的收缩反应;而Glib(10μmol/L）对其无明显影响。结论：Kv参与大鼠支气管平滑肌静息张力的调控,而BKCa和KATP对其无影响。Kv和BKCa的关闭增强组胺及高浓度钾离子诱发大鼠离体支气管产生的收缩张力。     

超氧阴离子通过提高[Ca2+]cyt调节保卫细胞K+通道和气孔运动

氧化信号参与了许多生理过程的调控。用膜片钳和激光共聚焦显微镜,采用可以产生O2^ 的甲基紫精处理蚕豆(Vicia faba L)保卫细胞,测定了O2^ 对气孔运动调节过程中胞质Ca^2 离子浓度和细胞质膜K^ 通道活性的变化,结果表明甲基紫精可以促进气孔的关闭,乙二醇四乙酸酯(Ethylene glycol bis(2-aminoethyl)tetra-acetic acid,EGTA)、抗坏血酸(Ascorbic acid,AsA)和过氧化物酶(Catalase,CAT)可以消除小于10^-5mol／L甲基紫精对气孔运动的影响;10^-2和10^-5mol/L的甲基紫精可使保卫细胞胞质Ca^2 浓度有不同程度提高,并伴随有钙震荡。蚕豆气孔保卫细胞质膜内向K^ 通道可被咆外甲基紫精抑制,而这种抑制和[Ca^2 ]cyt有关。推测甲基紫精产生的O2^-对蚕豆气孔运动的调节,主要是通过O2^ 诱导的胞内游离Ca^2 浓度的升高,从而抑制了通过保卫细胞质膜K^ 内向电流。     

Pronounced differences between the native K+ channels and KAT1 and KST1 α-subunit homomers of guard cells

Stomatal opening is the result of K⁺-salt accumulation in guard cells. Potassium uptake in these motor cells is mediated by voltage-dependent, K⁺-selective ion channels. Here we compare the in-vitro properties of two guard-cell K⁺-channel α-subunits from Arabidopsis thaliana (L.) Heynh. (KAT1) and Solanum tuberosum L. (KST1) after heterologous expression with the respective K⁺-transport characteristics in their mother cell. The KAT1 and KST1 subunits when expressed in Xenopus oocytes shared the basic features of the K⁺-uptake channels in the corresponding guard cells, including voltage dependence and single-channel conductance. Besides these similarities, the electrophysiological comparison of K⁺ channels in the homologous and the heterologous expression systems revealed pronounced differences with respect to modulation and block by extracellular cations. In the presence of 1 mM Cs⁺, 50% of the guard-cell K⁺-uptake channels (GCKC1_in) in A. thaliana and S. tuberosum, were inhibited upon hyperpolarization to −90 mV. For a similar effect on KAT1 and KST1 in oocytes, voltages as negative as −155 mV were required. In contrast, compared to the K⁺ channels in vivo the functional α-subunit homomers almost lacked a voltage-dependent block by extracellular Ca²⁺. Similar to the block by Cs⁺ and Ca²⁺, the acid activation of the α-homomers was less pronounced in oocytes. Upon acidification the voltage-dependence shifted by 82 and 90 mV for GCKCL_in in A. thaliana and S. tuberosum, respectively, but only by 25 mV for KAT1 and KST1. From the differences in K⁺-channel modulation in vivo and after heterologous expression we conclude that the properties of functional guard-cell K⁺-uptake channels result either from the heterometric assembly of different α-subunits or evolve from cell-type-specific posttranslational modification. Received: 6 March 1998 / Accepted: 9 July 1998     

Increases in cytosolic Ca2+ are not required for abscisic acid-inhibition of inward K+ currents in guard cells of Vicia faba L.

 The inward K⁺ channels (I_Kin) of guard cells are inhibited upon application of abscisic acid (ABA). It has been postulated that I_Kin inhibition requires an elevation in cytosolic free Ca²⁺ levels ([Ca²⁺]_c) because: (i) experimental increases in [Ca²⁺] _c can mimic the ABA effect, and; (ii) ABA can trigger an elevation of [Ca²⁺]_c in guard cells. However, not all guard cells respond to ABA with a [Ca²⁺]_c increase, and the magnitude of the increases that do occur is variable. Therefore, an obligate role for Ca²⁺ in the regulation of downstream effectors of ABA response, such as the I_Kin channels, remains in question. In this study, we developed a methodology for simultaneous patch clamping and confocal ratiometric Ca²⁺ imaging of Vicia faba L. guard-cell protoplasts. This allowed us to directly assess the relationship between ABA-induced changes in [Ca²⁺]_c and I_Kin inhibition. In the presence of extracellular Ca²⁺, the extent of [Ca²⁺]_c elevation correlated with the extent of I_Kin inhibition. However, upon chelation of either extracellular Ca²⁺, [Ca²⁺]_c, or both, extracellular Ca²⁺ and [Ca²⁺]_c, [Ca²⁺]_c elevation did not occur in response to ABA yet I_Kin currents were still strongly inhibited. These data illustrate that Ca²⁺-independent regulation is involved in ABA-inhibition of stomatal opening processes. Received: 17 September 1999 / Accepted: 26 October 1999     

Two-pore domain potassium channels: variation on a structural theme

The ability of cells to reliably fire action potentials is critically dependent upon the maintenance of a hyperpolarized resting potential, which allows voltage-gated Na(+) and Ca(2+) channels to recover from inactivation and open in response to a subsequent stimulus. Hodgkin and Huxley first recognized the functional importance a small, steady outward leak of K(+) ions to the resting potential, action potential generation and cellular excitability, and we now appreciate the contribution of inward rectifier-type K(+) channels (Kir or KCNJ channels) to this process. More recently, however, it has become evident that two-pore domain K(+) (K2P) channels also contribute to the steady outward leak of K(+) ions, and thus, maintenance of the resting potential. Molecular cloning efforts have demonstrated that K2P channel exist in yeast to humans, and represent a major branch in the K(+) channel superfamily. Humans express 15 types of K2P channels, which are grouped into six subfamilies, based on similarities in amino acid sequence and functional properties. Although K2P channels are not voltage-gated, due to the absence of a canonical voltage sensor domain, their activity can be regulated by a variety of stimuli, including mechanical force, polyunsaturated fatty acids (PUFAs) (e.g., arachidonic acid), volatile anesthetics, acidity/pH, pharmacologic agents, heat and signaling events, such as phosphorylation and protein-protein interactions. K2P channels thus represent important regulators of cellular excitability by virtue of their impact on the resting potential, and as such, have garnered considerable attention in recent years.     

Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes

Muscarinic receptor-linked G protein, G _i , can directely activate the specific K⁺ channel (I _K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G _i to the K⁺ channel in the ventricle has not been well defined. G protein regulation of K⁺ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization, mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I _K(ACh) and/or the background inwardly-rectifying K⁺ channel (I _K1) in ventricular cell membranes. I _K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I _K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I _K(ACh), G protein-activated I _K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I _K(ACh) and 1.2 μm in I _K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic receptor-linked PTX-sensitive G protein, G _i , is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action potential by activating I _K(ACh) and I _K1 via muscarinic receptor-linked G _i proteins in human ventricular myocytes. Received: 23 September 1996/Revised: 18 December 1996     

Charybdotoxin blocks both Ca-activated K channels and Ca-independent voltage-gated K channels in rat brain synaptosomes

Charybdotoxin (ChTX), a 4.3 kDa polypeptide toxin from the venom of the scorpion Leiurus quinquestriatus, blocks both a Ca-activated K channel (IC₅₀ ≈ 15 nM) and a Ca-independent voltage-gated K channel (IC₅₀ ≈ 40 nM) in rat brain synaptosomes. These results indicate that in this preparation ChTX is not specific for the Ca-activated K channel and suggest that there may be structural homology among the toxin-binding sites on various types of K channels.     

Stretch-activated chloride,potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L.

Mechanosensitive ion channels in the plasma membrane of Vicia faba guard cell protoplasts were studied by use of the patch clamp technique. Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics and ion selectivity. We found three distinct SA channels, permeable to Cl^–, K⁺ and Ca²⁺ and distinguishable from spontaneous (non-SA) channels for these ions on the basis of conductance, kinetics, and voltage-dependence, as well as sensitivity to membrane stretch. In the attached patch configuration, light suction (2 to 10 kPa) reversibly induced channel opening with multiple amplitudes and complex kinetics. The open probability for SA channels increased nonlinearly with pipette suction. In guard cells in situ, these SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and cytoplasmic calcium. Through such effects, SA channels likely influence volume and turgor regulation of guard cells, and thereby control of leaf gas exchange.Abbreviations EK equilibrium potential for potassium transport - E_Cl equilibrium potential for chloride transport - SA stretchactivated Dedicated to the 80. birthday of Franz HedrichSupported by a grant from the Deutsche Forschungsgemeinschaft to R.H. and a Department of Energy grant to D.J.C. gratefully acknowledges a John S. Guggenheim Fellowship and Fulbright Kommission Senior Professor Award. We thank Ingrid Baumann and Angela Schön for technical assistance, and Klaus Raschke and Heiner Busch for spirited discussions and support.     

Mitochondrial potassium channels and reactive oxygen species

Pretreatment of tissues with potassium channel openers (KCO’s) has been observed to be cytoprotective in a broad variety of insults. This phenomenon has been proposed to be intimately linked to activation of mitochondrial potassium channels which apparently modulate the mitochondrial production of reactive oxygen species (ROS). This critical review summarizes literature findings about the mitochondrial production of ROS, the action of KCO’s on mitochondrial ROS production and the putative link to the cytoprotective action of these drugs.     

The pore structure and gating mechanism of K2P channels

Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K(+) channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing.     

Protein phosphorylation is required for diazoxide to open ATP-sensitive potassium channels in insulin (RINm5F) secreting cells

The patch-clamp open-cell recording configuration has been used to investigate the effects of non-hydrolyzable analogues of ATP on the diazoxide-activation of K_ATP channels in the insulin-secreting cell line RINm5F. K⁺ channels inhibited by 0.1, 0.5 and 1.0 mM ATP were consistently activated by 200 μM diazoxide. During sustained activation of channels, exchange of ATP for either AMP-PNP, AMP-PCP or ATPγS abolished the effects of diazoxide. If diazoxide was added to the membrane in the continued presence of AMP-PNP, AMP-PCP or ATPγS either no effects were observed or alternatively a small transient activation of channels occurred. This study suggests that protein phosphorylation is necessary for diazoxide to activate ATP-sensitive potassium channels in insulin-secreting cells.     

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K⁺ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K⁺ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the K_m(PEP) and V_max values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM Crassulacean acid metabolism - GCP guard-cell protoplast - PEP phosphoenolpyruvate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Implication that potassium flux and increase in intracellular calcium are necessary for the initiation of sperm motility in salmonid fishes

Flux of K⁺ and changes in intracellular Ca²⁺ in the sperm of salmonid fishes were measured with spectrophotometry, ion electrode, microscopic fluorometry, and radioisotope accumulation. Release of K⁺ occurred at the initiation of sperm motility which is induced by decrease in external K⁺ and the K⁺ efflux and sperm motility were inhibited by K⁺ channel blockers. Intracellular Ca²⁺ increased within a short period in K⁺- free condition, and the accumulation of ⁴⁵Ca in sperm cells was higher in motile sperm than that in immotile sperm. The efflux of K⁺ and the increase in intracellular Ca²⁺ were suppressed when external K⁺ concentration increased, i.e., sperm remained immotile. These results suggest that efflux of K⁺ through K⁺ channel and subseqent increase in intracellular Ca²⁺ are prerequisite for the initiation of sperm motility. © 1994 Wiley-Liss, Inc.     

Extracellular calcium is involved in stomatal movement through the regulation of water channels in broad bean

Involvement of extracellular Ca²⁺ in stomatal movement through the regulation of water channels was investigated in broad bean (Vicia faba L.). Leaf peels were first incubated to open stomata, and then transferred to buffers in the presence of different CaCl₂ concentrations. Stomatal status was observed under magnification and stomatal aperture (pore width/length) was measured. Stomatal closure was significantly induced and aperture oscillation occurred at lower extracellular concentrations of calcium ([Ca²⁺]_ext), while at higher concentrations, no significant change in stomatal aperture was observed, which was similar to the response recorded with HgCl₂. Lower [Ca²⁺]_ext-induced stomatal closure could be reversed using depolarizing buffer. It is suggested that lower [Ca²⁺]_ext regulates water channels through an indirect way and at higher concentrations, extracellular Ca²⁺ is involved in regulating stomatal aperture by directly influencing water channels to retard aperture change.     

Coupling of activation and inactivation gate in a K+‐channel: potassium and ligand sensitivity

Potassium (K⁺)‐channel gating is choreographed by a complex interplay between external stimuli, K⁺ concentration and lipidic environment. We combined solid‐state NMR and electrophysiological experiments on a chimeric KcsA–Kv1.3 channel to delineate K⁺, pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH‐induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K⁺ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K⁺ ion on the inactivation gate modulate activation‐gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K⁺‐channel pore.     

Volume stabilization in human erythrocytes: combined effects of Ca-dependent potassium channels and adenylate metabolism

A mathematical model describing the possible role of Ca²⁺-dependent K⁺ channels and adenylate metabolism in volume stabilization of human erythrocytes was developed. The model predicts that the red blood cell volume can be stabilized either dynamically or stationary over a broad range of cell membrane permeabilities to cations. The dynamic stabilization results from the operation of Ca²⁺-dependent potassium channels. The erythrocyte volume changes less than 10% if the membrane permeability changes abruptly to a value in the range from half to sevenfold higher than the normal one. The stationary stabilization is achieved via controlling the adenylate metabolism. The stationary value of cell volume changes less than 10% when the membrane permeability varies from half the normal value to 15-fold higher than the normal value.     

Susceptibility of the guard-cell K+-uptake channel KST1 to Zn2+ requires histidine residues in the S3-S4 linker and in the channel pore

 Potassium channels are inhibited by several mono- and divalent cations. To identify sites involved in the interaction between K⁺ channels and cationic effectors, we expressed the potato (Solanum tuberosum L.) guard-cell K⁺-uptake channel KST1 in Xenopus oocytes. This channel was reversibly blocked by extracellular Zn²⁺ in the micromolar range. In the presence of this heavy metal, steady-state currents were reduced in a pH-dependent but voltage-independent manner. Since Zn²⁺-inhibition was less effective at elevated external proton concentrations, we generated alanine mutants with respect to both extracellular histidines in KST1. Whereas substitution of the pore histidine H271 resulted in a reduced blockade by Zn²⁺, the channel mutant KST1-H160A in the S3-S4 linker lost most of its Zn²⁺ sensitivity. Since both histidines alter the susceptibility of KST1 to Zn²⁺, the block may predominantly result from these two sites. We thus conclude that the S3-S4 linker is involved in the formation of the outer pore. Received: 3 May 1999 / Accepted: 8 July 1999