A laser microsurgical method of cell wall removal allows detection of largeconductance ion channels in the guard cell plasma membrane

Summary Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts ofVicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. “Laserassisted” patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.     

Local Anesthetics Affect Gramicidin A Channels via Membrane Electrostatic Potentials

The effects of local anesthetics (LAs), including aminoamides and aminoesters, on the characteristics of single gramicidin A (GA) channels in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers were studied. Aminoamides, namely lidocaine (LDC), prilocaine (PLC), mepivacaine (MPV), and bupivacaine (BPV), reduced the conductance of GA channels. Aminoesters influenced the current fluctuations induced by GA differently; procaine (PC) did not affect the fluctuations, whereas tetracaine (TTC) distinctly reduced the conductance of single GA channels. Using electrophysiological technique, we estimated the changes in the membrane boundary potential at the adsorption of LAs; LDC, PLC, MPV, BPV, and TTC substantially increased, while PC did not affect it. To elucidate which component of the membrane boundary potential, the surface or dipole potential, is responsible for the observed effects of LAs, we employed a fluorescence assay. We found that TTC led to a significant increase in the membrane dipole potential, whereas the adsorption of LDC, PLC, MPV, BPV, and PC did not produce any changes in the membrane dipole potential. We concluded that aminoamides affected the surface potential of lipid bilayers. Together, these data suggest that the effects of LAs on the conductance of single GA channels are caused by their influence on membrane electrostatic potentials; the regulation of GA pores by aminoamides is associated with the surface potential of membranes, whereas TTC modulation of channel properties is predominantly due to changes in dipole potential of lipid bilayers. These data might provide some significant implications for voltage-gated ion channels of cell membranes.     

A laser microsurgical method of cell wall removal allows detection of largeconductance ion channels in the guard cell plasma membrane

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.     

血管内皮细胞的Cl-通道--电特性和有关细胞功能

内皮细胞在心血管系统具有重要功能,除通过分泌内皮舒张因子－－一氧化氮（ＮＯ）及收缩性物质内皮素等控制血管平滑肌张力外,并能调节血管通透性。近年来发现内皮细胞上的Ｃ１＾－通道能调节细胞体积和细胞膜电位的稳定性。通过离子通道调控膜电位一机理,能较好理解血管内皮的功能,并可望由此开拓新型血管药物。本文综述了内皮细胞的Ｃ１＾－通道的电生理特性、类别,并探讨该通道调控细胞体积,ＮＯ的分泌及调控细胞膜电位的可     

A novel Cl- inward-rectifying current in the plasma membrane of the calcifying marine phytoplankton Coccolithus pelagicus

We investigated the membrane properties and dominant ionic conductances in the plasma membrane of the calcifying marine phytoplankton Coccolithus pelagicus using the patch-clamp technique. Whole-cell recordings obtained from decalcified cells revealed a dominant anion conductance in response to membrane hyperpolarization. Ion substitution showed that the anion channels were selective for Cl(-) and Br(-) over other anions, and the sensitivity to the stilbene derivative 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, ethacrynic acid, and Zn(2+) revealed a pharmacological profile typical of many plant and animal anion channels. Voltage activation and kinetic characteristics of the C. pelagicus Cl(-) channel are consistent with a novel function in plants as the inward rectifier that tightly regulates membrane potential. Membrane depolarization gave rise to nonselective cation currents and in some cases evoked action potential currents. We propose that these major ion conductances play an essential role in membrane voltage regulation that relates to the unique transport physiology of these calcifying phytoplankton.     

植物细胞质膜离子通道研究进展

多种有机和无机离子作为重要的营养物质、渗透物质、辅酶和信号分子, 参与植物生殖、生长发育和逆境反应等多种生物学过程。离子通道是离子跨质膜和内膜运动的重要渠道和动态调控因子, 直接影响和调控细胞内离子浓度及亚细胞分布的动态变化。目前, 植物尤其是模式植物拟南芥(Arabidopsis thaliana)的多个离子通道家族被先后鉴定出来, 其中部分离子通道蛋白定位在细胞质膜上, 其基本生物学功能, 诸如蛋白结构、离子选择性和通透性、门控特点、活性调控机理以及不同离子通道之间的协同关系等均取得重要进展。该文概要介绍近年来植物细胞质膜离子通道方面的研究进展。     

Effects of membrane lipids on ion channel structure and function

Biologic membranes are not simply inert physical barriers, but complex and dynamic environments that affect membrane protein structure and function. Residing within these environments, ion channels control the flux of ions across the membrane through conformational changes that allow transient ion flux through a central pore. These conformational changes may be modulated by changes in transmembrane electrochemical potential, the binding of small ligands or other proteins, or changes in the local lipid environment. Ion channels play fundamental roles in cellular function and, in higher eukaryotes, are the primary means of intercellular signaling, especially between excitable cells such as neurons. The focus of this review is to examine how the composition of the bilayer affects ion channel structure and function. This is an important consideration because the bilayer composition varies greatly in different cell types and in different organellar membranes. Even within a membrane, the lipid composition differs between the inner and outer leaflets, and the composition within a given leaflet is both heterogeneous and highly dynamic. Differential packing of lipids (and proteins) leads to the formation of microdomains, and lateral diffusion of these microdomains or "lipid rafts" serve as mobile platforms for the clustering and organization of bilayer constituents including ion channels. The structure and function of these channels are sensitive to specific chemical interactions with neighboring components of the membrane and also to the biophysical properties of their membrane microenvironment (e.g., fluidity, lateral pressure profile, and bilayer thickness). As specific examples, we have focused on the K+ ion channels and the ligand-gated nicotinicoid receptors, two classes of ion channels that have been well-characterized structurally and functionally. The responsiveness of these ion channels to changes in the lipid environment illustrate how ion channels, and more generally, any membrane protein, may be regulated via cellular control of membrane composition.     

植物质膜钾离子转运体研究进展

近年,随着分子生物学技术的不断发展和广泛应用,有关植物质膜钾离子转运体的研究取得重要进展。目前已经克隆到多种质膜钾离子转运体基因并对钾离子转运体生化特性以及结构功能进行了广泛研究。研究认为,质膜钾离子转运体可分为钾离子载体和钾离子通道。钾离子通道又可分为内向性K+通道α亚基、K+通道β亚基及外向性K+通道等三类。本文对上述质膜钾离子转运体的生化特性以及结构功能研究的进展进行了综述。     

The hidden potential of lysosomal ion channels: A new era of oncogenes

Lysosomes serve as the control centre for cellular clearance. These membrane-bound organelles receive biomolecules destined for degradation from intracellular and extracellular pathways; thus, facilitating the production of energy and shaping the fate of the cell. At the base of their functionality are the lysosomal ion channels which mediate the function of the lysosome through the modulation of ion influx and efflux. Ion channels form pores in the membrane of lysosomes and allow the passage of ions, a seemingly simple task which harbours the potential of overthrowing the cell’s stability. Considered the master regulators of ion homeostasis, these integral membrane proteins enable the proper operation of the lysosome. Defects in the structure or function of these ion channels lead to the development of lysosomal storage diseases, neurodegenerative diseases and cancer. Although more than 50 years have passed since their discovery, lysosomes are not yet fully understood, with their ion channels being even less well characterized. However, significant improvements have been made in the development of drugs targeted against these ion channels as a means of combating diseases. In this review, we will examine how Ca²⁺, K⁺, Na⁺ and Cl⁻ ion channels affect the function of the lysosome, their involvement in hereditary and spontaneous diseases, and current ion channel-based therapies.     

Potassium and its role in cesium transport in plants

In plant cells, potassium (K⁺) is abundantly present and is dominant cation plays a vital role in maintaining physiological and morphological characteristics of plants. Many membrane integrated channels and transporters specific to K⁺ are involved in maintaining the potassium concentration within plants via membrane electrical activities. Elemental homologues to K⁺ compete with it for entry inside plants; among those, cesium is very common radionuclide. Once cesium enters into the plant cell, it can cause phytotoxicity. Therefore, it is desirable to understand complete pathway and mechanisms of cesium uptake in the plants, in order to assess consequences from accidental release of radioactive substance. This review focuses on mechanism of K⁺ ion uptake through channels/transporter and involvement of these channels/transporter in cesium uptake in plant cells.     

Fast and slow activation of voltage-dependent ion channels in radish vacuoles.

The molecular processes associated with voltage-dependent opening and closing (gating) of ion channels were investigated using a new preparation from plant cells, i.e., voltage and calcium-activated ion channels in radish root vacuoles. These channels display a main single channel conductance of approximately 90 pS and are characterized by long activation times lasting several hundreds of milliseconds. Here, we demonstrate that these channels have a second kinetically distinct activation mode which is characterized by even longer activation times. Different membrane potential protocols allowed to switch between the fast and the slow mode in a controlled and reversible manner. At transmembrane potentials of -100 mV, the ratio between the fast and slow activation time constant was around 1:5. Correspondingly, activation times lasting several seconds were observed in the slow mode. The molecular process controlling fast and slow activation may represent an effective modulator of voltage-dependent gating of ion channels in other plant and animal systems.     

Modelisation of the contribution of the Na/Ca exchanger to cell membrane potential and intracellular ion concentrations

Modelisation plays a significant role in the study of ion transfer through the cell membrane and in the comprehension of cellular excitability. We were interested in the selective ion transfers through the K(Ca), Na(v), Ca(v) channels and the Na/Ca exchanger (NCX). The membrane behaves like an electric circuit because of the existence of ion gradients maintained by the cell. The non-linearity of this circuit gives rise to complex oscillations of the membrane potential. By application of the finite difference method (FDM) and the concept of percolation we studied the role of the NCX in the regulation of the intracellular Ca(2+) concentration and the oscillations of the membrane potential. The fractal representation of the distribution of active channels allows us to follow the diffusion of intracellular Ca(2+) ions. These calculations show that the hyperpolarization and the change in the burst duration of the membrane potential are primarily due to the NCX.     

Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel

Cyclic nucleotide-gated (cng) non-selective cation channels have been cloned from a number of animal systems. These channels are characterized by direct gating upon cAMP or cGMP binding to the intracellular portion of the channel protein, which leads to an increase in channel conductance. Animal cng channels are involved in signal transduction systems; they translate stimulus-induced changes in cytosolic cyclic nucleotide into altered cell membrane potential and/or cation flux as part of a signal cascade pathway. Putative plant homologs of animal cng channels have been identified. However, functional characterization (i.e. demonstration of cyclic-nucleotide-dependent ion currents) of a plant cng channel has not yet been accomplished. We report the cloning and first functional characterization of a plant member of this family of ion channels. The Arabidopsis cDNA AtCNGC2 encodes a polypeptide with deduced homology to the alpha-subunit of animal channels, and facilitates cyclic nucleotide-dependent cation currents upon expression in a number of heterologous systems. AtCNGC2 expression in a yeast mutant lacking a low-affinity K(+) uptake system complements growth inhibition only when lipophilic cyclic nucleotides are present in the culture medium. Voltage clamp analysis indicates that Xenopus laevis oocytes injected with AtCNGC2 cRNA demonstrate cyclic-nucleotide-dependent, inward-rectifying K(+) currents. Human embryonic kidney cells (HEK293) transfected with AtCNGC2 cDNA demonstrate increased permeability to Ca(2+) only in the presence of lipophilic cyclic nucleotides. The evidence presented here supports the functional classification of AtCNGC2 as a cyclic-nucleotide-gated cation channel, and presents the first direct evidence (to our knowledge) identifying a plant member of this ion channel family.     

Plasma Membrane-Bound Mechanisms of Signal Transduction in the Control of Plant Dormancy and Resistance

Data on membrane-bound biochemical mechanisms of control of plant dormancy and resistance to phytopathogens are discussed. Phytohormones are involved in the control of plant dormancy by the modification of activity of membrane-bound enzymes and ion channels. Similar constituents of the plasma membrane are influenced by fungal extracellular metabolites. Proposed interconnections between plasmalemma-bound signaling mechanisms responsible for plant resistance to infection and dormancy regulation are illustrated by a scheme.     

Characterization of the Variation Potential in Sunflower

A major candidate for intercellular signaling in higher plants is the stimulus-induced systemic change in membrane potential known as variation potential (VP). We investigated the mechanism of occurrence and long-distance propagation of VP in sunflower (Helianthus annuus L.) plants. Here we present evidence of the relationship among injury-induced changes in xylem tension, turgor pressure, and electrical potential. Although locally applied wounding did trigger a change in membrane potential, it evoked even faster changes in tissue deformation, apparently resulting from pressure surges rapidly transmitted through the xylem and experienced throughout the plant. Externally applied pressure mimicked flame wounding by triggering an electrical response resembling VP. Our findings suggest that VP in sunflower is not a propagating change in electrical potential and not the consequence of chemicals transmitted via the xylem, affecting ligand-modulated ion channels. Instead, VP appears to result from the surge in pressure in the xylem causing a change in activity of mechanosensitive, stretch-responsive ion channels or pumps in adjacent, living cells. The ensuing ion flux evokes local plasma membrane depolarization, which is monitored extracellularly as VP.     

Ion channels and transporters regulate nutrient absorption in health and disease

Ion channels and transporters are ubiquitously expressed on cell membrane, which involve in a plethora of physiological process such as contraction, neurotransmission, secretion and so on. Ion channels and transporters is of great importance to maintaining membrane potential homeostasis, which is essential to absorption of nutrients in gastrointestinal tract. Most of nutrients are electrogenic and require ion channels and transporters to absorb. This review summarizes the latest research on the role of ion channels and transporters in regulating nutrient uptake such as K+ channels, Ca2+ channels and ion exchangers. Revealing the mechanism of ion channels and transporters associated with nutrient uptake will be helpful to provide new methods to diagnosis and find potential targets for diseases like diabetes, inflammatory bowel diseases, etc. Even though some of study still remain ambiguous and in early stage, we believe that ion channels and transporters will be novel therapeutic targets in the future.     

Membrane potential and time requirements for detection of weak signals by voltage‐gated ion channels

The question of minimum detection limits for biological processes sensitive to membrane potential perturbations has arisen in various contexts. Of special interest are the prediction of theoretical limits for sensory perception processes and for possible biological effects of environmental or therapeutic electric and magnetic fields. A new method is presented here, addressing the particular case in which perturbations of membrane potential affect the gating rate probability of voltage‐sensitive ion channels. Using a two‐state model for channel gating, the influence of the perturbing potential on the mean fraction of open channels is approximated by a Boltzmann distribution, and integrated over time to obtain a quantity proportional to the net change in expected charge transfer through the membrane. This change in net charge transfer (the signal, S) is compared to the expected root mean variance in charge transfer (the noise, N) due to random channel gating. Using a nominal criterion of S/N = 1, a model is developed for predicting the minimum time and number of ion channels necessary to detect a given membrane potential. Example calculations, carried out for a gating charge of 6, indicate that a 1 μV induced membrane potential can be detected after 10 ms by an ensemble of less than 10⁸ ion channels. Bioelectromagnetics 20:102–109, 1999. Published 1999 Wiley‐Liss, Inc.     

Zipper transition in an alpha-helix: a mechanism for gating of voltage-sensitive ion channels in a biological membrane

It is suggested that the gating currents which control the ion channels in a biological membrane are comprised of positive charges crossing the membrane along chains of hydrogen bonds. These chains are the sets of hydrogen bonds which hold alpha-helical protein segments in their rigid conformations. The passage of a positive charge in one direction along such a chain will convert hydrogen bonds from the usual rigid N--C = O...H--N form to a flaccid N = C--O--H...N form. This "zipper" transition can be reversed by the passage of the positive charge along the return route. A flaccid protein rod can clog an ion channel and thereby close it. When all of the protein rods framing an ion channel are in the rigid conformation, the channel is open. This mechanism is used to explain some of the observed characteristics of calcium ion channels and sodium ion channels.     

Glutamate receptors in plants

Ionotropic glutamate receptors function in animals as glutamate-gated non-selective cation channels. Numerous glutamate receptor-like (GLR) genes have been identified in plant genomes, and plant GLRs are predicted, on the basis of sequence homology, to retain ligand-binding and ion channel activity. Non-selective cation channels are ubiquitous in plant membranes and may function in nutrient uptake, signalling and intra-plant transport. However, there is little evidence for amino acid gating of plant ion channels. Recent evidence suggests that plant GLRs do encode non-selective cation channels, but that these channels are not gated by amino acids. The functional properties of these proteins and their roles in plant physiology remain a mystery. The problems surrounding characterization and assignation of function to plant GLRs are discussed in this Botanical Briefing, and potential roles for GLR proteins as non-selective cation channels involved in metabolic signalling are described.     

Ion channels in nerve ending

The modern data about the structure and function of the nerve ending ion channels are generalized and systematized. Ion channels of nerve endings provide the forming of the rest membrane potential, excitability, generation of action potential, regulate the intracellular concentration of calcium ions, take part in exocytosis of synaptic vesicules, participate in short-term and long-term synaptic plasticity, ensure the modulation of presynaptic functions. Methods of investigation of ion channels and data about their localization in central and peripheral nerve systems are represented. The review gives the functional characteristics, molecular structure and mechanisms of regulation of the known voltage- and ligand-dependent ion channels, the role of the certain types of ion channels in the machinery of transmitter release.