Ionic channels,ion transport and plant cell membranes: Potential applications of the patch-clamp technique

Conclusion Exciting innovations in the methodologies available for the study of ionic channels (notably in animal cells) have allowed hitherto impossible advances in the comprehension of both structure and function. In using channels like the Na channel and the AChR as examples of these strategies, we have tried to give a concise but up to date account of the current possibilities (in particular, the patch-clamp) for research in membrane physiology. That few of these techniques have been applied to plant cell membranes simply indicates the scope for advancement in the understanding of some problems fundamental to plant physiology. The mechanisms of transport involved in processes known to be important for the life of plant cells (e.g., regulation of cytoplasmic and vacuolar potential differences and pH, maintenance of vacuolar turgor pressure, accumulation of metabolites and their counterions, response to environmental stimuli) are relatively speaking, poorly characterized. In that ion fluxes through plasmalemma and tonoplast membranes are at least in part likely to be via ionic channels for all of these processes, an important step forward would be the application of patch-clamp techniques for the direct demonstration of a channel mechanism and the subsequent elucidation of their role.     

细胞电生理技术在昆虫抗药性研究中的应用

害虫几乎对所有化学农药及Bt等生物农药都产生了抗性。离子通道是多种杀虫剂的作用靶,因此作为研究离子通道基本手段的电压钳与膜片钳技术在害虫抗性检测与抗性机理研究中越来越受到重视。该文综述了细胞电生理技术在害虫抗性机理、杀虫剂作用机理以及药物筛选中的应用。     

Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence

Ion channels and transporters are membrane proteins whose functions are driven by conformational changes. Classical biophysical techniques provide insight into either the structure or the function of these proteins, but a full understanding of their behavior requires a correlation of both these aspects in time. Patch-clamp and voltage-clamp fluorometry combine spectroscopic and electrophysiological techniques to simultaneously detect conformational changes and ionic currents across the membrane. Since its introduction, patch-clamp fluorometry has been responsible for invaluable advances in our knowledge of ion channel biophysics. Over the years, the technique has been applied to many different ion channel families to address several biophysical questions with a variety of spectroscopic approaches and electrophysiological configurations. This review illustrates the strength and the flexibility of patch-clamp fluorometry, demonstrating its potential as a tool for future research.     

Multiple Regeneration Pathways via Thin Cell Layers in Hybrid Cymbidium (Orchidaceae)

Protocorm-like body (PLB) and subsequent shoot development in hybrid Cymbidium Twilight Moon ‘Day Light’ can be established in vitro via 3 pathways: PLBs, PLB thin cell layers (TCLs), or embryogenic callus (EC). Traditionally Cymbidium hybrids are mass-produced commercially through the neo-formation of secondary PLBs (2° PLB) from initial or primary PLBs (1° PLB) or PLB segments, or from PLB TCLs, resulting in a moderate number of 2° PLBs (average 4.46 2° PLBs/1° bisected PLB, or 1.12 2° PLBs/ PLB TCL). This study shows that EC can be induced from 1° PLBs or PLB TCLs. Thereafter, resulting 2° PLBs (average 22.1 2° PLBs/EC cluster derived from 1° PLB) form directly from the EC on the same medium or following the transfer of EC onto PGR-free medium. By flow cytometry and PCR-RAPD analysis, the cytogenetic stability of 1° PLBs, of resulting 2° PLBs and EC, and plants derived therefrom was demonstrated.     

Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence

Ion channels and transporters are membrane proteins whose functions are driven by conformational changes. Classical biophysical techniques provide insight into either the structure or the function of these proteins, but a full understanding of their behavior requires a correlation of both these aspects in time. Patch-clamp and voltage-clamp fluorometry combine spectroscopic and electrophysiological techniques to simultaneously detect conformational changes and ionic currents across the membrane. Since its introduction, patch-clamp fluorometry has been responsible for invaluable advances in our knowledge of ion channel biophysics. Over the years, the technique has been applied to many different ion channel families to address several biophysical questions with a variety of spectroscopic approaches and electrophysiological configurations. This review illustrates the strength and the flexibility of patch-clamp fluorometry, demonstrating its potential as a tool for future research.     

The role of ion channels in plant nutrition and prospects for their genetic manipulation

Ion channels can function in three physiological modes through their ability to: 1) accommodate osmotically significant fluxes over short periods; 2) propagate signals along or across membranes; 3) control the membrane potential. With respect to mineral nutrition it is via the control of the membrane potential that ion channels are probably most significant. In this paper the physiology and prospects for molecular biology of plant ion channels are discussed. It is concluded that identifying and altering the primary structures that determine functional characteristics of plant ion channel genes could result in changes in the transport characteristics of higher plants.     

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。     

In vitro propagation of <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchid through formation of protocorm-like bodies

Paphiopedilum orchids are among the world’s most popular orchid due to their impressively beautiful flowers. Propagation of these orchid genera has been hampered by the naturally slow growth rate of the plant, which renders it very difficult to be propagated through conventional methods. In vitro culture techniques have provided a useful alternative technology for propagating this recalcitrant species. In this study, the propagation of P. rothschildianum was achieved through the in vitro formation of secondary protocorm-like bodies (PLBs) from the primary PLB that developed from stem-derived callus. The PLBs were cultured on half-strength MS medium supplemented with different concentrations (1.0, 2.0, 3.0, and 4.0 μM) of 6-benzyladenine (BA) and kinetin for the induction of secondary PLBs. The highest number of secondary PLBs formed was obtained on half-strength MS medium supplemented with 4.0 μM kinetin, with an average of 4.1 PLBs per explant after 8 weeks of culture. The secondary PLBs continued to proliferate further and formed 9.5–12.1 new PLBs per secondary PLB after being subcultured onto half-strength plant growth regulator-free MS medium supplemented with 60 g/L banana homogenate (BH). These tertiary PLBs were subcultured onto media containing different organic additives, such as BH, coconut water, potato homogenate, and tomato homogenate, for plantlet regeneration. Among the organic additives tested, the addition of 20% CW to half-strength MS medium resulted in the best average plantlet regeneration percentage from the PLBs, 67.9%, after 8 weeks of culture.     

Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development

The prolamellar body (PLB) proteome of dark-grown wheat leaves was characterized. PLBs are formed not only in etioplasts but also in chloroplasts in young developing leaves during the night, yet their function is not fully understood. Highly purified PLBs were prepared from 7-day-old dark-grown leaves and identified by their spectral properties as revealed by low-temperature fluorescence spectroscopy. The PLB preparation had no contamination of extra-plastidal proteins, and only two envelope proteins were found. The PLB proteome was analysed by a combination of 1-D SDS-PAGE and nano-LC FTICR MS. The identification of chlorophyll synthase in the PLB fraction is the first time this enzyme protein was found in extracts of dark-grown plants. This finding is in agreement with its previous localization to PLBs using activity studies. NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyses the reduction of protochlorophyllide to chlorophyllide, dominates the proteome of PLBs. Besides the identification of the PORA protein, the PORB protein was identified for the first time in dark-grown wheat. Altogether 64 unique proteins, representing pigment biosynthesis, photosynthetic light reaction, Calvin cycle proteins, chaperones and protein synthesis, were identified. The in number of proteins’ largest group was the one involved in photosynthetic light reactions. This fact strengthens the assumption that the PLB membranes are precursors to the thylakoids and used for the formation of the photosynthetic membranes during greening. The present work is important to enhance our understanding of the significance of PLBs in chloroplast development.     

Evidence for protein phosphatase 1 and 2A regulation of K+ channels in two types of leaf cells.

Ion channels control ion fluxes across membranes, membrane potential, and signal transduction between and within cells. Protein kinases and phosphatases are important regulators involved in stimulus-response coupling in eukaryotic organisms. We have identified in extracts of Vicia faba leaf cells protein phosphatase activities inhibited by okadaic acid (OA) and calyculin A (CA), two inhibitors of protein phosphatases 1 and 2A. Using whole-cell patch-clamp techniques, we have demonstrated that inward K+ currents in guard cells are inhibited by nanomolar concentrations of OA or CA, whereas outward K+ currents are not affected. However, the same inhibitors enhance the magnitude of outward K+ currents in mesophyll cells. A phosphatase antagonist, adenosine-5'-O-(3-thiotriphosphate), has an effect similar to OA and CA on outward K+ currents in mesophyll cells. Our findings suggest that protein phosphatases 1 and/or 2A play different physiological roles in modulating the activity of K+ channels in mesophyll cells and guard cells.     

Insights from modeling three-dimensional structures of the human potassium and sodium channels

Ion channels allow the movement of ions across cell membranes. Nearly all cells have membranes spanned by ion channels, without which human nerves simply would not work. Ion channels are formed by the aggregation of subunits into a cylindrical configuration that allows a pore, thus forming a kind of tube for ion trafficking. In the present study, the subunits of the human potassium channel are formed by four identical protein chains, whereas for the case of the human sodium channel, the corresponding subunits are actually four hetero-domains formed by the folding of a very large but single protein chain. Since both of the two ion channels are important targets for drug discovery, the 3D (dimensional) structures of their pore regions were developed. On the basis of the 3D models, some important molecular biological mechanisms were discussed that may stimulate novel strategies for therapeutic treatment of the diseases related to ion channel disorders, such as long QT syndrome and chronic pain.     

Visualization and characterization of prolamellar bodies with atomic force microscopy

Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1–2 μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1 μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25 min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl₂ also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.     

Separation of K+-and Cl --selective ion channels from rye roots on a continuous sucrose density gradient

Microsomal membranes from rye (Secale cereale L.) roots wereseparated by isopycnic sucrose density gradient centrifugation.The ion channels present in gradient fractions were assayedby reconstitution into planar 1-palmitoyl-2-oleoyl phosphatidylethanolaminebilayers (PLB) and the distributions of ion channel activitieswere compared with membrane markerenzyme activities. A numberof ion channel activities were observed and could be distinguishedon the combined bases of their conductance, selectivity, kineticsand pharmacology. A voltage-dependent maxi (498 pS) cation-channel,a voltage-dependent 199-pS cationchannel, 48-pS and 18-pS K⁺channels, and a 148-pS Cl^– channel (all unitary conductancesdetermined in asymmetrical cis trans 325:100mM KCl) colocalizedwith the plasma membrane marker-enzyme, vanadatesensitive ATPase.A weakly K ⁺-selective (108 pS) channel, a 1249-pS cation-channeland a 98-pS K ⁺ channel colocalized with the tonoplast markerenzyme,nitrate-sensitive ATPase. A 706-pS K⁺ channel colocalized withthe expected distribution of intact plastids and a 38-pS Cl^–channel colocalized with either plastid or ER membranes. Themembrane location of several other channels including a hypervoltage-sensitivemaxi (497 pS) cation-channel, a 270-pS K⁺ channel, an 8-pS K⁺channel and a 4-pS K⁺ channel was equivocal, but they were tentativelyassigned to the Golgi. Thus, the plasma membrane and tonoplastorigin of ion channels previously characterized following theincorporation of plasma membrane prepared by aqueous-polymertwo-phase partitioning or tonoplast derived from isolated vacuolesinto PLB was confirmed and the ion channel complement of previouslyunassayed membranes was defined. This demonstrates the usefulnessof PLB in identifying and characterizing ion channels from plantcell membranes, in particular, those of membranes which areinaccessible to patch-clamp electrodes. Key words: Chloride (Cl^–) channel, potassium (K⁺) channel, planar lipid bilayer, root, rye, Secale cerealeL.     

结直肠离子通道与先天性巨结肠关系的研究进展

离子通道是细胞膜上一类特殊亲水性蛋白微孔道,也是肌肉、神经细胞等电活动的物质基础。目前研究通过生物学及离子通道膜片钳等新技术对离子通道有了进一步的认识,并逐步发掘离子通道的结构功能异常与疾病的发生存在的紧密关系。先天性巨结肠症(Hirschsprung's Disease,HD)又称无神经节细胞症,是小儿外科的常见疾病之一。HD临床表现为胎粪排出延迟、顽固性便秘及腹胀,常并发小肠结肠炎、低位肠梗阻等。目前研究尚未完全明确HD的发病机制,本文对HD的发生与结直肠离子通道功能间的关系作一综述。     

Distinct ion channel classes are expressed on the outer nuclear envelope of T- and B-lymphocyte cell lines

The outer nuclear membrane, endoplasmic reticulum, and mitochondrial membrane ion channels are poorly understood, although they are important in the control of compartmental calcium levels, cell division, and apoptosis. Few direct recordings of these ion channels have been made because of the difficulty of accessing these intracellular membranes. Using patch-clamp techniques on isolated nuclei, we measured distinct ion channel classes on the outer nuclear envelope of T-cell (human Jurkat) and BFL5 cell (murine promyelocyte) lines. We first imaged the nuclear envelopes of both Jurkat and FL5 cells with atomic force microscopy to determine the density of pore proteins. The nuclear pore complex was intact at roughly similar densities in both cell types. In patch-clamp recordings of Jurkat nuclear membranes, Cl channels (105 +/- 5 pS) predominated and inactivated with negative pipette potentials. Nucleotides transiently inhibited the anion channel. In contrast, FL5 nuclear channels were cation selective (52 +/- 2 pS), were inactivated with positive membrane potentials, and were insensitive to GTPgammaS applied to the bath. We hypothesize that T- and B-cell nuclear membrane channels are distinct, and that this is perhaps related to their unique roles in the immune system.     

Priming biotic factors for optimal protocorm-like body and callus induction in hybrid Cymbidium (Orchidaceae), and assessment of cytogenetic stability in regenerated plantlets

Protocorm-like bodies (PLBs) and callus were induced in epiphytic hybrid Cymbidium Twilight Moon ‘Day Light’, where induction capacity was strongly explant dependent. Following the use of various explant sources (PLB, leaf tip or base, root tip or base, cell and tissue ‘suspension’), highest PLB formation and callus induction occurred when we used whole PLBs, PLB segments or PLB transverse thin cell layers (tTCLs) or longitudinal TCLs (lTCLs). Plantlet growth and photosynthetic state from whole or bisected PLBs, as well as from tTCLs were not significantly different, after analysis of chlorophyll content. However plantlets generated from lTCLs showed lower values for growth and photosynthetic parameters. All resultant plants were shown to be cytogenetically identical using RAPD and mtDNA analysis despite cytological variation and endopolyploidy occuring between different plant parts. Acclimatization and survival rate was shown to be 100% in the generated plants.     

Novel screening techniques for ion channel targeting drugs

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.     

Shoot development and plant regeneration from protocorm-like bodies of <Emphasis Type="Italic">Zygopetalum mackayi</Emphasis>

Stem nodal segments of a sympodial orchid, Zygopetalum mackayi, were used as explants to induce protocorm-like body (PLB) formation on a hormone-free 1/2 Murashige and Skoog (1962) modified medium (1/2MS-0) or 1/2MS supplemented with 0.045–4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ] in light. After 1 mo of culture, pale to dark green, compact and irregular nodulars of PLBs formed from the explants. For PLB induction, TDZ had no significant effect on the percentage of PLB formation but promoted mean numbers of PLBs per responding explant at 0.045–4.54 μM. For plant conversion, PLBs were transferred onto the same basal medium devoid of TDZ. After 2–3 mo of culture, these PLBs successfully formed shoots and then roots with normal morphology. For PLB proliferation, TDZ has no significant effects on the fresh weight of PLB aggregates, but there is significantly retarded shoot development at 0.45–4.54 μM after 1 mo of culture. When transferring these PLB aggregates onto hormone-free medium for plant conversion, PLBs derived from TDZ-containing medium showed a decrease of shoot length (0.86–2.08 cm in shoot length) compared to those derived from 1/2MS-0 (2.74 cm in shoot length) after 1 mo of culture. Gibberellin A₃ [GA₃] at 0.29–8.66 μM significantly retarded PLB proliferation, but at 0.03 and 0.29 μM resulted in longer shoot length than the control treatment. Histological studies reveal that shoot development originated from the outer region of PLB aggregates. The young shoots initially connected to each other at their basal tissues with the parental PLBs. Plants were successfully obtained from PLBs and then gradually became more loosely connected with each other as well as with the parental aggregates. Several dozen plants were acclimatized in the greenhouse and showed normal morphology.     

An efficient direct induction of protocorm-like bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture

High-frequency protocorm-like body (PLB) formation directly from thin leaf sections of Doritaenopsis hybrid was achieved in order to develop a mass-scale propagation system. Concentrated efforts were made to study the effects of different cytokinins on in vitro PLB induction from thin leaf sections. Among the cytokinins tested, thidiazuron (TDZ) was found to be a more effective inducer of PLBs than benzyladenine and zeatin. A modified Murashige and Skoog medium supplemented with 9.0 µM TDZ was found to be the optimum concentration for PLB development from thin leaf sections of Doritaenopsis hybrid. Of the two different explant types used in the present experiment, the highest percentage of PLB formation (72.3%) and highest number of PLBs (18) per explant were observed on thin leaf sections (1 mm thick), while only 20% (4.3 per explant) of comparatively large leaf segments (5 mm thick) were able to produce PLBs under the same culture conditions. Light microscopy observations indicated that the initial cell divisions for PLB formation occurred on the region near the cut surface and that an intact epidermal layer appeared to play an important role in PLB formation. Proembryo initiation occurred from several cells just beneath the intact epidermal cell, and globular PLBs were clearly visible after 3 weeks of culture and subsequently developed into mature PLBs.     

Outward-Rectifying K+ Channels in Stomatal Guard Cell Protoplasts

Ion channels in stomatal guard cell protoplasts from Vicia fabawere examined using the patch-clamp technique. Most ion channelshaving unit conductance ranging between 10 and 30 pS showedclear outward-rectification in symmetrical 50mM KCl. The largeinside-out membranes contained these outward-rectifiers as themajor and relatively stable channels. The channels were K⁺ ion-selective.Kinetic analysis revealed that the channels have three conductancestates: open, closed and inactivated. The rates of transitionto and from the inactivated state were highly voltage-dependent. (Received April 6, 1988; Accepted May 25, 1988)