Determining K+ channel activation curves from K+ channel currents

Potassium ion channels are generally believed to have current-voltage (IV) relations which are linearly related to driving force ( V - E(K)), where V is membrane potential and E(K) is the potassium ion equilibrium potential. Consequently, activation curves for K+ channels have often been measured by normalizing voltage-clamp families of macroscopic K+ currents with (V - E(K)), where V is the potential of each successive step in the voltage clamp sequence. However, the IV relation for many types of K+ channels actually has a non-linear dependence upon driving force which is well described by the Goldman-Hodgkin-Katz relation. When the GHK dependence on (V - E(K)) is used in the normalization procedure, a very different voltage dependence of the activation curve is obtained which may more accurately reflect this feature of channel gating. Novel insights into the voltage dependence of the rapidly inactivating I(A) channels Kv1.4 and Kv4.2 have been obtained when this procedure was applied to recently published results.     

K+ channel activity in plants: genes, regulations and functions

Potassium (K(+)) is the most abundant cation in the cytosol, and plant growth requires that large amounts of K(+) are transported from the soil to the growing organs. K(+) uptake and fluxes within the plant are mediated by several families of transporters and channels. Here, we describe the different families of K(+)-selective channels that have been identified in plants, the so-called Shaker, TPK and Kir-like channels, and what is known so far on their regulations and physiological functions in the plant.     

Regulation by arbuscular mycorrhizae of the integrated physiological response to salinity in plants: new challenges in physiological and molecular studies

Excessive salt accumulation in soils is a major ecological and agronomical problem, in particular in arid and semi-arid areas. Excessive soil salinity affects the establishment, development, and growth of plants, resulting in important losses in productivity. Plants have evolved biochemical and molecular mechanisms that may act in a concerted manner and constitute the integrated physiological response to soil salinity. These include the synthesis and accumulation of compatible solutes to avoid cell dehydration and maintain root water uptake, the regulation of ion homeostasis to control ion uptake by roots, compartmentation and transport into shoots, the fine regulation of water uptake and distribution to plant tissues by the action of aquaporins, the reduction of oxidative damage through improved antioxidant capacity and the maintenance of photosynthesis at values adequate for plant growth. Arbuscular mycorrhizal (AM) symbiosis can help the host plants to cope with the detrimental effects of high soil salinity. There is evidence that AM symbiosis affects and regulates several of the above mentioned mechanisms, but the molecular bases of such effects are almost completely unknown. This review summarizes current knowledge about the effects of AM symbiosis on these physiological mechanisms, emphasizing new perspectives and challenges in physiological and molecular studies on salt-stress alleviation by AM symbiosis.     

Five-group distribution of the Shaker-like K+ channel family in higher plants

Abstract In higher plants, potassium channels of the Shaker family have been shown to play crucial roles in the uptake of K⁺ from the soil solution and subsequent transport of this ion at the cell, tissue, and organ levels. In the model plant Arabidopsis thaliana, this family is composed of nine members, which are the best characterized among plant channels at the protein, gene, and functional property levels. Plant Shaker channels share a common structure: a hydrophobic core composed of six transmembrane segments, a long cytoplasmic C-terminal region harboring a putative cyclic nucleotide binding domain, and a K_HA domain. Many channels also contain an ankyrin domain between the putative cyclic nucleotide binding domain and the K_HA domain. The analysis of 44 Shaker channels from plants revealed a five-group classification. The members of each group share high sequence and structure similarities. This grouping also correlates with the diversification of the functional properties of the proteins, as members of an individual group have roughly the same electrophysiological characteristics. Analysis of the intron positions showed that the gene structures are also quite well conserved within the five groups. A correlation linking the evolution of the sequences and the positioning of the introns was established. Finally, a moss sequence provided additional clues about the hypothetical structure of an ancestor of the present channels and suggested that the diversification of plant Shaker channels happened before the separation of monocots and dicots and after the separation of bryophytes and tracheophytes.     

Regulation of physiological aspects in plants by hydrogen sulfide and nitric oxide under challenging environment

Plants are exposed to a plethora of abiotic stresses such as drought, salinity, heavy metal and temperature stresses at different stages of their life cycle, from germination to seedling till the reproductive phase. As protective mechanisms, plants release signaling molecules that initiate a cascade of stress-signaling events, leading either to programmed cell death or plant acclimation. Hydrogen sulfide (H₂S) and nitric oxide (NO) are considered as new ‘gasotransmitter’ molecules that play key roles in regulating gene expression, posttranslational modification (PTM), as well as cross-talk with other hormones. Although the exact role of NO in plants remains unclear and is species dependent, various studies have suggested a positive correlation between NO accumulation and environmental stress in plants. These molecules are also involved in a large array of stress responses and act synergistically or antagonistically as signaling components, depending on their respective concentration. This study provides a comprehensive update on the signaling interplay between H₂S and NO in the regulation of various physiological processes under multiple abiotic stresses, modes of action and effects of exogenous application of these two molecules under drought, salt, heat and heavy metal stresses. However, the complete picture of the signaling cascades mediated by H₂S and NO is still elusive. Recent researches indicate that during certain plant processes, such as stomatal closure, H₂S could act upstream of NO signaling or downstream of NO in response to abiotic stresses by improving antioxidant activity in most plant species. In addition, PTMs of antioxidative pathways by these two molecules are also discussed.     

Electrophysiological analysis of structural aspects of voltage-dependent SR K+ channel.

This article presents a brief review on the electrophysiological analysis of the structural aspects of the voltage-dependent SR (sarcoplasmic reticulum) K+ channel. In the first half, early attempts to determine the physical dimensions of the ion conducting mechanism such as the mouth, narrow tunnel, or ion selective filter of the channel, are reviewed. The depicted cartoon of the SR K+ channel, as an extremely short, busy district with a big mouth on each side, is quite similar to the recently-obtained reconstructed structural image of the acetylcholine receptor channel. In the latter half, we introduce our recent attempts to draw a physical image of the gating mechanism of the SR K+ channel. We examined, for example, the location of the gate and the voltage sensor, and the relationship between them. It is suggested that the gate and the sensor are connected tightly and that the sensor would be exposed to the surface of the lumen side of SR when the gate opens. Finally, the issue of substates in SR K+ channel is discussed. It is implied that the substrate-conductances reflect a partial occlusion of the pore by an intermediate-open gate.     

Towards an understanding of the molecular basis of plants K+ transport: Characterization of cloned K+ transport cDNAs

Recently, two K⁺-transport cDNAs, KAT1 and AKT1, were cloned in Arabidopsis thaliana. These cDNAs had structural similarities to K⁺ channel genes in animals, and also conferred the ability for growth on micromolar levels of K⁺ when expressed in K⁺ transport-defective yeast mutants. In this study, we examined the possibility that KAT1 encodes the high-affinity K⁺ transport system that has been previously characterized in plant roots, by studying the concentration-dependent kinetics of K⁺ transport for KAT1 expressed in Xenopus oocytes and Saccharomyces cerevisiae. In both organisms, the K⁺ transport system encoded by KAT1 yielded Michaelis-Menten kinetics with a high K_m for K⁺ (35 mM in oocytes, 0.6 mM in yeast cells). Furthermore, Northern analysis indicated that KAT1 is expressed primarily in the Arabidopsis shoot. These results strongly suggest that the system encoded by KAT1 is not a root high-affinity K⁺ transporter.     

Plant cell responses to heavy metals: molecular and physiological aspects

The effect of lead, cadmium and cooper on protein pattern, free radicals and antioxidant enzymes in root of Lupinus luteus L. were investigated. Heavy metals inhibited growth of lupin roots, which was accompanied by increased synthesis and accumulation of a 16 kDa polypeptide (Przymusiński et al. 1991 Biochem. Physiol. Pflanzen., 187:51–57). This component has been earlier identified as immunologically related to Cu,Zn-superoxide dismutase (Przymusiński et al. 1995 Env.Exp.Bot., 35:485–495). However, more detailed study revealed that this stress-stimulated protein is composed of four to six polypeptides of different electrophoretic mobility. The most abundant polypeptides of the 16kDa region were found to be closely homologous to pathogen related proteins. The number and intensity of these polypeptides was highly variable in roots of individual seedlings, which suggests that they might represent separate allelic forms. Electron paramagnetic spectra revealed that at low lead concentrations the amplitude of the first derivative was similar to the control and distinctly increased at higher metal concentrations. On the other hand, at the lower lead concentrations the activity of antioxidant enzymes increased, whereas at higher metal doses the enzyme activities did not raise further (SOD) or even dropped (CAT, APOX). This implies that the responses of antioxidant system to lead is dose-dependent stimulated by low metal concentrations, whereas at the higher metal level the free radical emission is beyond the quenching capacity of antioxidant enzymes, which in turn might contribute to the reduced root growth. The effect of various heavy metals: Pb²⁺, Cd²⁺ and Cu²⁺ on phytochelatins and antioxidant enzymes depends on the kind of metal ion. Pb²⁺ and Cd²⁺ stimulated the PCs formation whereas Cu²⁺ was not effective. On the other hand, in root exposed to Cu the activity of catalase (CAT) was the highest as was the production of H₂O₂. The strong oxidative effect of Cu²⁺ ions which were not complexed by PCs suggests that these peptides might by involved in the cellular defense system by binding excessive heavy metal ions. On the basis of our results it can be concluded that in lupin roots exposed to heavy metals there is a complex defense system against metal phytotoxicity, which comprises of specific proteins, antioxidant enzymes and phytochelatins.     

Conduction of Na+ and K+ through the NaK channel: molecular and Brownian dynamics studies

Conduction of ions through the NaK channel, with M0 helix removed, was studied using both Brownian dynamics and molecular dynamics. Brownian dynamics simulations predict that the truncated NaK has approximately a third of the conductance of the related KcsA K⁺ channel, is outwardly rectifying, and has a Michaelis-Menten current-concentration relationship. Current magnitude increases when the glutamine residue located near the intracellular gate is replaced with a glutamate residue. The channel is blocked by extracellular Ca²⁺. Molecular dynamics simulations show that, under the influence of a strong applied potential, both Na⁺ and K⁺ move across the selectivity filter, although conduction rates for Na⁺ ions are somewhat lower. The mechanism of conduction of Na⁺ differs significantly from that of K⁺ in that Na⁺ is preferentially coordinated by single planes of pore-lining carbonyl oxygens, instead of two planes as in the usual K⁺ binding sites. The water-containing filter pocket resulting from a single change in the selectivity filter sequence (compared to potassium channels) disrupts several of the planes of carbonyl oxygens, and thus reduces the filter's ability to discriminate against sodium.     

Intracellular K+ sensing of SKOR, a Shaker-type K+ channel from Arabidopsis

Most K+ channels in plants are structurally classified into the Shaker family named after the shaker K+ channel in Drosophila. Plant K+ channels function in many physiological processes including osmotic regulation and K+ nutrition. An outwardly rectifying K+ channel, SKOR, mediates the delivery of K+ from stelar cells to the xylem in the roots, a critical step in the long-distance distribution of K+ from roots to the upper parts of the plant. Here we report that SKOR channel activity is strictly dependent on intracellular K+ concentrations. Activation by K+ did not affect the kinetics of voltage dependence in SKOR, indicating that a voltage-independent gating mechanism underlies the K+ sensing process. Further analysis showed that the C-terminal non-transmembrane region of the SKOR protein was required for this sensing process. The intracellular K+ sensing mechanism couples SKOR activity to K+ nutrition status in the 'source cells', thereby establishing a supply-based unloading system for the regulation of K+ distribution.     

Ca2+-Activated K+ channel from human erythrocyte membranes: Single channel recification and selectivity

Summary The Ca²⁺-activated K⁺ channel of the human red cell membranes was characterized with respect to rectification and selectivity using the patch-clamp technique. In inside-out patches exposed to symmetric solutions of K⁺, Rb⁺, and NH ₄ ⁺ , respectively, inward rectifyingi-V curves were obtained. The zero current conductances were: K⁺ (23.5 pS±3.2)>NH ₄ ⁺ (14.2 pS±1.2)>Rb⁺ (11.4 pS±1.8). With low extracellular K⁺ concentrations (substitution with Na⁺) the current fluctuations reversed close to the Nernst potential for the K ion and the rectification as well as thei-V slopes decreased. With mixed intracellular solutions of K⁺ and Na⁺ enhanced rectification were observed due to a Na⁺ block of outward currents. From bi-ionic reversal potentials the following permeability sequence (P _K/P _X) was calculated: K⁺ (1.0)>Rb⁺ (1.4±0.1)>NH ₄ ⁺ (8.5±1.3)>Li⁺(>50); Na⁺ (>110); Cs⁺ (5). Li⁺, Na⁺, and Cs⁺ were not found to carry any current, and only minimum values of the permeability ratios were estimated. Tl⁺ was permeant, but the permeability and conductance were difficult to quantify, since with this ion the single channel activity was extremely low and the channels seemed to inactivate. The inward rectification in symmetric solutions indicate an asymmetric open channel structure, and the different selectivity sequences based on conductances and permeabilities reflect interionic interactions in the permeation process.     

Single channel K+ currents from HeLa cells

The extracellular patch-clamp technique was used in order to investigate the presence of ionic channels in HeLa cells, a well-known cultured cell type obtained from an epidermoid carcinoma of the cervix. Under Gigohm-seal conditions, discrete current jumps could be observed with patch electrodes containing KCl. These channels were found to be mainly permeable to K+ and showed multiple levels of conductance. From single-channel I-V curve measurements, a strong rectification effect, characterized by a large inward and no detectable outward current, was observed. For negative membrane potentials (0 to -90 mV), the measured current-voltage relationship was found to be mostly linear, corresponding to a single-channel conductance of 40 pS. An analysis of some selected time records has revealed in addition that the probability of the channel to be in the open state was a function of the KCl concentration in the patch pipette.     

几种木本植物的N2O释放与某些生理活动的关系

使用带有开放气路的气体交换测定系统,同步测定了几种针、阔叶树种的光合作用、呼吸作用及气孔导度．结果表明,低光下树木针叶或叶片释放Ｎ２Ｏ的速率与光合速率无显著相关．伴随根、茎、叶的呼吸,检测到有Ｎ２Ｏ吸收现象,其通量与温度及呼吸强度呈正相关．气孔导度明显影响Ｎ２Ｏ的通量,表明气孔可能是木本植物释放Ｎ２Ｏ的主要途径．     

亚高寒草甸植物叶片生理指标对坡向的响应

研究了甘南亚高寒草甸不同坡向条件下矮嵩草、狼毒和棘豆叶片的叶绿素、游离脯氨酸和可溶性糖含量,以及稳定碳同位素(δ¹³C)的变化,分析干旱胁迫条件下,植物适应干旱胁迫的生理机制.结果表明: 随着坡向由北坡-西北坡-西坡-西南坡到南坡的变化,土壤含水量(北坡0.36 g·g^-1,南坡0.15 g·g^-1)呈降低趋势,土壤温度(北坡14.76 ℃,南坡24.85 ℃)和光照度(北坡540.34 lx,南坡744.12 lx)呈增加趋势;植物物种的组成也随之发生了变化,北坡主要有灌木金露梅及杂类草,而南坡主要有禾草类物种.3种植物叶片的脯氨酸、可溶性糖、叶绿素含量及稳定碳同位素(δ¹³C)随着坡向的变化均有不同程度的变化,且物种不同,各物种的生理指标变化幅度也有差异.在坡向梯度上,3种植物的脯氨酸、可溶性糖含量和稳定碳同位素与土壤含水量均呈显著负相关,与温度和光照强度呈显著正相关;植物叶片叶绿素与土壤含水量呈显著正相关,与温度和光照强度呈显著负相关.其中,土壤含水量是坡向梯度上影响植物生长的关键因子.植物叶片生理指标(脯氨酸、可溶性糖及叶绿素等)可以作为衡量植物抗逆性的因素,3种植物的抗性大小顺序为:矮嵩草>狼毒>棘豆.     

Tethered blockers as molecular 'tape measures' for a voltage-gated K+ channel

The propagation of electrical signals in excitable cells is orchestrated by a molecular family of voltage-dependent ion channel proteins. These K+, Na+, and Ca++ channels are all composed of four identical or similar units, each containing six transmembrane segments (S1-S6) in a roughly four-fold symmetric structure. The S5-S6 sequences fold into a central pore unit, which is surrounded by a voltage-gating module composed of S1-S4. The recent structure of KcsA, a two-transmembrane bacterial K+ channel, illuminates the physical character of the pore unit, but little is known about the arrangement of the surrounding S1-S4 sequences. To locate regions of this gating module in space, we synthesized a series of compounds of varying length that function as molecular 'tape measures': quaternary ammonium (QA) pore blockers that can be tethered to specific test residues. We show that in a Shaker K+ channel, the extracellular ends of S1 and S3 are approximately 30 ? from the tetraethylammonium (TEA) blocking site at the external opening of the pore. A portion of the S3-S4 loop is, at 17-18 ?, considerably closer.