Heteromeric K+ channels in plants

Voltage-gated potassium channels of plants are multimeric proteins built of four α-subunits. In the model plant Arabidopsis thaliana , nine genes coding for K⁺ channel α-subunits have been identified. When co-expressed in heterologous expression systems, most of them display the ability to form heteromeric K⁺ channels. Till now it was not clear whether plants use this potential of heteromerization to increase the functional diversity of potassium channels. Here, we designed an experimental approach employing different transgenic plant lines that allowed us to prove the existence of heteromeric K⁺ channels in plants. The chosen strategy might also be useful for investigating the activity and function of other multimeric channel proteins like, for instance, cyclic-nucleotide gated channels, tandem-pore K⁺ channels and glutamate receptor channels.     

ATP-regulated K+ channel in mitochondria: Pharmacology and function

Mitochondria from several tissues contain a potassium-specific channel similar to the ATP-regulated K⁺ (K_ATP) channel of the plasma membrane. The mitochondrial channel shares with the plasma membrane K_ATP channel the sensitivity to sulfonylurea derivatives and some other blockers as well as to channel openers of diverse chemical character. In contrast to the plasma membrane channel, which is blocked by free ATP, the mitochondrial K_ATP channel reconstituted into liposomes requires the ATP-Mg complex for inhibition. The mitochondrial K_ATP channel, possibly in a concerted action with other K⁺ permeability pathways, plays an important role in mitochondrial volume control. Its function in the regulation of the components of the protonmotive force is also suggested.     

Shab K+ channel slow inactivation

Herein, we report the first characterization of Shab slow inactivation. Open Shab channels inactivate within seconds, with two voltage-independent time constants. Additionally, Shab presents significant closed-state inactivation. We found that with short depolarizing pulses, shorter than the slowest inactivation time constant, the resulting inactivation curve has a marked U-shape, but as pulse duration increases, approaching steady-state conditions, the U-shape vanishes, and the resulting inactivation curves converge to the classical Boltzmann h_∞ curve. Regarding the mechanism of inactivation, we found that external K⁺ and TEA facilitate both open- and closed-state inactivation, while the cavity blocker quinidine hinders inactivation. These results together with our previous observations regarding the K⁺-dependent stability of the K⁺ conductance, suggest the novel hypothesis that inactivation of Shab channels, and possibly that of other Kv channels whose inactivation is facilitated by K⁺, does not involve a significant narrowing of the extracellular entry of the pore. Instead, we hypothesize that there is only a rearrangement of a more internal segment of the pore that affects the central cavity and halts K⁺ conduction.     

Root high-affinity K+ and Cs+ uptake and plant fertility in tomato plants are dependent on the activity of the high-affinity K+ transporter SlHAK5

Root K⁺ acquisition is a key process for plant growth and development, extensively studied in the model plant Arabidopsis thaliana. Because important differences may exist among species, translational research supported by specific studies is needed in crops such as tomato. Here we present a reverse genetics study to demonstrate the role of the SlHAK5 K⁺ transporter in tomato K⁺ nutrition, Cs⁺ accumulation and its fertility. slhak5 KO lines, generated by CRISPR-Cas edition, were characterized in growth experiments, Rb⁺ and Cs⁺ uptake tests and root cells K⁺-induced plasma membrane depolarizations. Pollen viability and its K⁺ accumulation capacity were estimated by using the K⁺-sensitive dye Ion Potassium Green 4. SlHAK5 is the major system for high-affinity root K⁺ uptake required for plant growth at low K⁺, even in the presence of salinity. It also constitutes a pathway for Cs⁺ entry in tomato plants with a strong impact on fruit Cs⁺ accumulation. SlHAK5 also contributes to pollen K⁺ uptake and viability and its absence produces almost seedless fruits. Knowledge gained into SlHAK5 can serve as a model for other crops with fleshy fruits and it can help to generate tools to develop low Cs⁺ or seedless fruits crops.     

Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels

To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 microM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 +/- 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 +/- 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2-5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an "IQ-like" motif present in the carboxy terminus of KCNQ2-5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic "gene gun." Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor.     

Inhibition of Transient K+ Current by Copper in Drosophila Neurons

The transient K+ current (IK(A)) affects the rate of repetitive action potentials. The whole-cell patch-clamp technique was applied to cultured Drosophila neurons derived from embryonic neuroblasts. IK(A) was measured from neurons before and after application of 0.1 mM copper to the external saline. IK(A) was smaller in the copper-containing saline (12.0 +/- 1.6 pA) than in the control saline (37 +/- 6.5 pA). Activation and inactivation of IK(A) were unchanged by copper. These results suggest that copper can influence neuronal excitability and may affect neuronal function.     

Construction of a cyclic nucleotide-gated KcsA K+ channel

The ability of an ion channel to open in response to a defined stimulus is central to its function. In ligand-gated channels, pore opening is conferred through transduction of a conformational change in a gating domain to the helices of the pore. Here, we present the construction of a designed cyclic nucleotide-gated (CNG) channel, named KcsA-CNG, by addition of a prokaryotic cyclic nucleotide-binding domain to a KcsA-derived K+ channel. This channel is functional in lipid bilayers at physiological pH and has the combined properties of both of its parent-derived components. It conducts K+ and is blocked by the K+ channel inhibitors Na+ and agitoxin-2. Channel open times are increased by about two orders of magnitude compared to wild-type KcsA. The average number of open channels increases by approximately 50% upon addition of cAMP. Although the absolute open probabilities are somewhat variable from one channel to the next, the property of cyclic nucleotide sensitivity is very reproducible. An apparent Kd value of approximately 90 nM was estimated. The successful construction of a cyclic nucleotide-gated KcsA K+ channel suggests that it should be possible to produce channels that will respond to novel ligands.     

Response of greenhouse tomato to salt stress and K+ supplement

Abstract

The effect of NaCl salinity and potassium supplement on growth, tissue ion concentration, photosynthesis, yield and fruit quality characteristics of tomato plants was studied. Tomato plants, hyb. Belladonna, were grown in 8.5 l pots, filled with 1:3 sand:perlite mixture and irrigated with a half-strength Hoagland solution through a closed hydroponic system. Six irrigation treatments were applied, including combinations of 3 salinity (0, 35 and 70 mM NaCl) and two potassium levels (K1: 200 ppm and K2: 400 ppm) in the nutrient solution. Salinity reduced photosynthesis resulting in reduced plant height and dry weight. Yield was reduced by 25% and 69% at 35 and 70 mM, respectively, as compared to control plants (0 mM NaCl). Both total soluble solids and titratable acidity of the fruit increased with increasing salinity and K levels. The application of high potassium level (K2) reduced the concentration of Na and increased that of K in the leaves and roots of the plants, as compared to K1 treatment. Toxicity symptoms were mostly observed in the leaves of 70K1 plants, while no visual symptoms of toxicity were observed in 70K2 treatment. Despite the positive effects of potassium supplement in reducing Na concentration and the absence of toxicity symptoms in the leaves, plant growth was not improved, while leaf photosynthesis was reduced. Furthermore, no positive effects in the percentage of marketable fruit, mean fruit weight and yield were observed in the plants receiving extra K.     

Ca2+-activated K+ currents inNecturus choroid plexus

Summary The tight-seal whole-cell recording method has been used to studyNecturus choroid plexus epithelium. A cell potential of –59±2 mV and a whole cell resistance of 56±6 M were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110mm NaCl Ringer solution and the interior of the cell contained a solution of 110mm KCl and 5nm Ca²⁺, stepping the membrane potential from a holding value of –50 to –10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca²⁺-activated K^_ channels. Based on the voltage dependence of the activation of Ca²⁺-activated K⁺ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K⁺ as their reversal potential at different K⁺ concentration gradients followed the Nernst potential for K⁺. These currents were reduced by the addition of TEA⁺ to the bath solution and were eliminated when Cs⁺ or Na⁺ replaced intracellular K⁺. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t _1/2) to the steady value. Increasing the pipette Ca²⁺ also decreased the delay and decreasedt _1/2. For instance, when pipette Ca²⁺ was increased from 5 to 500nm, the delay andt _1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K⁺ currents through Ca²⁺-activated K⁺ channels.At the resting membrane potential of –60 mV, Ca²⁺-activated K⁺ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20–30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca²⁺. Thus the Ca²⁺-activated K⁺ channels may play a specific role in maintaining intracellular K⁺ concentrations during CSF secretion.     

Calcium-activated K+ channels: Metabolic regulation

Calcium-activated potassium (K_Ca) channels are highly modulated by a large spectrum of metabolites. Neurotransmitters, hormones, lipids, and nucleotides are capable of activating and/or inhibiting K_Ca channels. Studies from the last few years have shown that metabolites modulate the activity of K_Ca channels via: (1) a change in the affinity of the channel for Ca²⁺ (K_1/2 is modified), (2) a parallel shift in the voltage axis of the acitvation curves, or (3) a change in the slope (effective valence) of the voltage dependence curve. The shift of the voltage dependence curve can be a direct consequence of the change in the affinity for Ca²⁺. Recently, the mechanistic steps involved in the modulation of K_Ca channels are being uncovered. Some interactions may be direct on K_Ca channels and others may be mediated via G-proteins, second messengers, or phosphorylation. The information given in this review highlights the possibility that K_Ca channels can be activated or inhibited by metabolites without a change in the intracellular Ca²⁺ concentration.     

Single-file diffusion through K+ channels in frog skin epithelium

Summary The ratio between the unidirectional fluxes of K⁺ across the frog skin with K-permeable outer membranes was determined in the absence of Na⁺ in the apical solutions. The experiments were performed under presteady-state conditions to be able to separate the flux ratio for K⁺ through the cells from contributions to the fluxes through extracellular leaks. The cellular flux ratio deviated strongly from the value calculated from the flux ratio for electrodiffusion. The experiments can be explained if the passive K transport through the epithelial cells proceeds through specific channels by single-file diffusion with a flux ratio exponent of about 2.5.     

The high affinity K+ transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis

Caesium (Cs⁺) is a potentially toxic mineral element that isreleased into the environment and taken up by plants. AlthoughCs⁺ is chemically similar to potassium (K⁺), and much is knownabout K⁺ transport mechanisms, it is not clear through whichK⁺ transport mechanisms Cs⁺ is taken up by plant roots. In thisstudy, the role of AtHAK5 in high affinity K⁺ and Cs⁺ uptakewas characterized. It is demonstrated that AtHAK5 is localizedto the plasma membrane under conditions of K⁺ deprivation, whenit is expressed. Growth analysis showed that AtHAK5 plays arole during severe K⁺ deprivation. Under K⁺-deficient conditionsin the presence of Cs⁺, Arabidopsis seedlings lacking AtHAK5had increased inhibition of root growth and lower Cs⁺ accumulation,and significantly higher leaf chlorophyll concentrations thanwild type. These data indicate that, in addition to transportingK⁺ in planta, AtHAK5 also transports Cs⁺. Further experimentsshowed that AtHAK5 mediated Cs⁺ uptake into yeast cells andthat, although the K⁺ deficiency-induced expression of AtHAK5was inhibited by low concentrations of NH     

Binding of kappa-conotoxin PVIIA to Shaker K+ channels reveals different K+ and Rb+ occupancies within the ion channel pore

The x-ray structure of the KcsA channel at different [K(+)] and [Rb(+)] provided insight into how K(+) channels might achieve high selectivity and high K(+) transit rates and showed marked differences between the occupancies of the two ions within the ion channel pore. In this study, the binding of kappa-conotoxin PVIIA (kappa-PVIIA) to Shaker K(+) channel in the presence of K(+) and Rb(+) was investigated. It is demonstrated that the complex results obtained were largely rationalized by differences in selectivity filter occupancy of this 6TM channels as predicted from the structural work on KcsA. kappa-PVIIA inhibition of the Shaker K(+) channel differs in the closed and open state. When K(+) is the only permeant ion, increasing extracellular [K(+)] decreases kappa-PVIIA affinity for closed channels by decreasing the "on" binding rate, but has no effect on the block of open channels, which is influenced only by the intracellular [K(+)]. In contrast, extracellular [Rb(+)] affects both closed- and open-channel binding. As extracellular [Rb(+)] increases, (a) binding to the closed channel is slightly destabilized and acquires faster kinetics, and (b) open channel block is also destabilized and the lowest block seems to occur when the pore is likely filled only by Rb(+). These results suggest that the nature of the permeant ions determines both the occupancy and the location of the pore site from which they interact with kappa-PVIIA binding. Thus, our results suggest that the permeant ion(s) within a channel pore can determine its functional and pharmacological properties.     

尼古丁调节大鼠冠状动脉平滑肌细胞大电导钙激活钾通道

目的:研究尼古丁对Wistar大鼠冠状动脉平滑肌大电导钙激活钾通道(BKca)活性的抑制作用及其细胞信号转导机制。方法:8周雄性Wistar大鼠随机分为两组:生理盐水组和尼古丁组;分别予以生理盐水和尼古丁2mg/(kg.d)注射21 d,蛋白酶法分离冠状动脉血管平滑肌细胞,将两组平滑肌细胞分别以对氯苯硫基环腺苷酸(CPT-cAMP,100μmol/L)和佛司可林(forskolin,10μmol/L)干预,单通道膜片钳记录干预前后平滑肌细胞单通道电流的平均开放时间(To)、平均关闭时间(Tc)、平均开放概率(Po)。结果:CPT-cAMP和Forskolin均能显著延长生理盐水组大鼠BKca的平均开放时间,缩短平均关闭时间,增加通道开放概率(P均<0.01)。对尼古丁组BKca的To、Tc、Po均无明显影响。结论:尼古丁促使冠状动脉血管收缩的生理机制是通过抑制cAMP/PKA途径诱导的大电导钙激活钾通道活性增加实现的。     

Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca²⁺ activation site was modified using N-bromoacetamide (NBA). 1mm NBA in the bath solution, at 2.5 m Ca²⁺ reduces the open probability,P _o , of the channel to <0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca²⁺-sensitive, requiring 25mm Ca²⁺ to raiseP _o to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca²⁺ (1mm) protects the channels from modification. Also presented is a second class of Ca²⁺-activated K⁺ channels which are normally present in MTAL cells which open infrequently at 10 m Ca²⁺ (P _o =0.01) but have aP _o of 0.08 at 1mm Ca²⁺. We can conclude (i) that NBA modifies the channel by shifting Ca²⁺-sensitivity to very high Ca²⁺, (ii) that NBA acts on a site involved in Ca²⁺ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.     

Ca2+-dependent K+ channels from rat olfactory cilia characterized in planar lipid bilayers

Olfactory cilia contain cyclic nucleotide-gated and Ca2+-dependent Cl- conductances that underlie excitatory chemotransduction, and a Ca2+-dependent K+ (KCa) conductance, apparently involved in inhibitory transduction. Previous single-channel patch-clamp studies on olfactory cilia revealed four different KCas, with different conductances and kinetics. Here, we further characterized these channels in planar bilayers, where blockers could be properly tested. All four ciliary KCas were observed: The 16 pS channel, K0.5,Ca=40 microM and apamin-sensitive; the 30 and 50 pS channel, K0.5,Ca=59 microM, clotrimazole-sensitive and charybdotoxin-insensitive; the 60 pS channel, clotrimazole-sensitive and charybdotoxin-insensitive; and the 210 pS channel, K0.5,Ca=63 microM, blocked by charybdotoxin and iberiotoxin. The presence of the 16 and 210 pS channels was confirmed by immunoblotting.     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

Permeance of Cs+ and Rb+ through the inwardly rectifying K+ channel in guinea pig ventricular myocytes

Summary Inward currents carried by external Cs, Rb, NH₄ and K through theI _K1 channel were studied using a whole-cell voltage clamp technique. Cs, NH₄, and Rb currents could be recorded negative to –40 mV following depolarizing prepulses (0 mV and 200–1000 msec in duration). The current activation displayed an instantaneous component followed by a monoexponential increase () to a peak amplitude. Subsequent inactivation was fit by a single exponential, _i. With hyperpolarization, and _i decreasede-fold per 36 and 25 mV, respectively. In Ca-free external solutions (pipette [Mg]0.3mm), inactivation was absent, consistent with the hypothesis that inactivation represents time- and voltage-dependent block of Cs, NH₄, and Rb currents by external Ca. The inactivation and degree of steady-state block was greatest when Cs was the charge carrier, followed by NH₄, and then Rb. K currents, however, did not inactivate in the presence of Ca. Na and Li did not carry any significant current within the resolution of our recordings. Comparison ofpeak inward current ratios (I _x/I_K) as an index of permeability revealed a higher permeance of Cs (0.15), NH₄ (0.30), and Rb (0.51) relative to K (1.0) than that obtained by comparing thesteady-state current ratios (CsNH₄RbK0.010.060.211.0). At any given potential, was smaller the more permeant the cation. In the absence of depolarizing prepulses, the amplitude of was reduced. Divalent-free solutions did not significantly affect activatio in the presence of 0.3mm pipette [Mg]. When pipette [Mg] was buffered to 50 m, however, removal of external Ca and Mg lead to a four- to fivefold increase in Cs currents and loss of both time-dependent activation and inactivation (reversible upon repletion of external Ca).These results suggest that (i) permeability ratios forI _K1 should account for differences in the degree to which monovalent currents are blocked by extracellular Ca and (ii) extracellular or intracellular divalent cations contribute to the slow phase of activation which may represent either (a) the actual rate of Mg or Ca extrusion from the channel into the cell, a process which may be enhanced by repulsive interaction with the incoming permeant monovalent cation or (b) an intrinsic gating process that is strongly modulated by the permeant monovalent ion and divalent cations.