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.     

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.     

Detection of a large cation-selective channel in nuclear envelopes of avian erythrocytes.

To determine whether the nuclear envelope of eukaryotic cells has the capability to regulate ion fluxes, we have used the patch-clamp technique to detect ion channels in this membrane system. Since possible sites for ion channels in the nuclear envelope include not only the nuclear pores, but also both the inner and outer nuclear membranes, we have patched giant liposomes composed of phosphatidylcholine and nuclear envelope fragments isolated from mature avian erythrocytes. A large, cation-selective channel with a maximum conductance of approximately 800 pS in symmetrical 100 mM KCl was detected. This channel is a possible candidate for a nuclear pore.     

Large-Conductance Cation Channels in the Envelope of Nuclei from Rat Cerebral Cortex

Eucaryotic nuclei are surrounded by a double-membrane system enclosing a central cisterna which is continuous with the endoplasmic reticulum and serves as a calcium store for intracellular signaling. The envelope regulates protein and nucleic acid traffic between the nucleus and the cytoplasm via nuclear pores. These protein tunnels cross through both nuclear membranes and are permeable for large molecules. Surprisingly, patch clamp recordings from isolated nuclei of different cell species have revealed a high resistance of the envelope, enabling tight seals and the resolution of single ion channel activity. Here we present for the first time single-channel recordings from nuclei prepared from neuronal tissue. Nuclei isolated from rat cerebral cortex displayed spontaneous long-lasting large conductances in the nucleus-attached mode as well as in excised patches. The open times are in the range of seconds and channel activity increases with depolarization. The single-channel conductance in symmetrical K⁺ is 166 pS. The channels are selective for cations with P _K/P _Na= 2. They are neither permeable to, nor gated by Ca²⁺. Thus, neuronal tissue nuclei contain a large conductance ion channel selective for monovalent cations which may contribute to ionic homeostasis in the complex compartments surrounding these organelles. Received: 12 November 1996/Revised: 18 February 1997     

USING ATOMIC FORCE MICROSCOPY TO INVESTIGATE PATCH-CLAMPED NUCLEAR MEMBRANE

Nuclear patch clamp is an emerging research field that aims to disclose the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its function as an intracellular calcium store. The authors combined the patch clamp technique with atomic force microscopy (AFM) to investigate the structure—function relationship of NE. In principle, patch clamp currents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes that compose the NE. However, the role of the NPCs is still unclear because the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applied to link patch clamp currents to structure. The membrane patch was excised from the nuclear envelope and, after electrical evaluation, transferred from the patch pipette to a substrate. We could identify the native nuclear membrane patches with AFM at a lateral and a vertical resolution of 3nm and 0.1nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their functional identification in patch clamp experiments. However, we also show that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This possibility must be considered carefully in nuclear patch clamp experiments.     

Studying mechanosensitive ion channels with an automated patch clamp

Patch clamp electrophysiology is the main technique to study mechanosensitive ion channels (MSCs), however, conventional patch clamping is laborious and success and output depends on the skills of the operator. Even though automated patch systems solve these problems for other ion channels, they could not be applied to MSCs. Here, we report on activation and single channel analysis of a bacterial mechanosensitive ion channel using an automated patch clamp system. With the automated system, we could patch not only giant unilamellar liposomes but also giant Escherichia coli (E. coli) spheroplasts. The tension sensitivity and channel kinetics data obtained in the automated system were in good agreement with that obtained from the conventional patch clamp. The findings will pave the way to high throughput fundamental and drug screening studies on mechanosensitive ion channels.     

Identification of a nucleo-cytoplasmic ionic pathway by osmotic shock in isolated mouse liver nuclei

Summary The observation that the nuclear envelope outer mem brane contains ion channels raises the question of whether these conductances communicate between the cytosol and the nuclear envelope cisternae or between the cytosol and the cytoplasm. Failure to detect large, nonselective holes using the patch-clamp technique has led to the speculation that ion channels and nuclear pores are in fact the same. In this paper we present evidence that the ionic channel, recorded in isolated liver nuclei with the patch-clamp configura tion of “nucleus-attached,” spans the double membrane of the envelope, providing a direct contact between nucleoplasm and cytoplasm.     

Gating mechanism of the nuclear pore complex channel in isolated neonatal and adult mouse liver nuclei.

Several types of ionic channels on the outer membrane of the nuclear envelope communicate with the nuclear cisternae. These are distinct from nucleocytoplasmic pathways, the nuclear pores that span the double membrane of the envelope and are the route for RNA and protein traffic in the nucleus. Recent data indicate that the nuclear pores may also function as ion channels. The most probable candidate for nucleocytoplasmic ion flux is a 300-400 pS pathway observed in many nuclear preparations. Morphological and functional studies of nuclear envelope suggest a tight relationship between the large conductance channel and the pore complex. However, there is no direct evidence for gating of the nuclear pore or its ability to open and close as a conventional channel. This study shows that in liver nuclei isolated from newborn mouse, there is a substantial correspondence between the number of pores and the number of channels recorded during patch-clamp. This is not the case for adult nuclei. Although pore density is comparable, some nuclear cytoskeletal components, such as actin and nonmuscle myosin, show a significant increase in the adult preparation. Previous studies demonstrate the presence of these two proteins in association with the pore complex. Here we show that by using actin filament disrupter, we were able to increase the number of active channels in adult isolated nuclei. We suggest that a functional interaction between actin filaments and the nuclear pore complex could regulate nucleocytoplasmic permeability.     

Cell age-related differences in the interaction of a potential-sensitive fluorescent dye with nuclear envelopes of Acetabularia mediterranea

Abstract. To study whether an electrical potential difference exists across the nuclear envelope or inner nuclear membrane of plant cells, the authors have used an optical probe of membrane potential, the cationic fluorescent dye, DiOC₆(3) (MW = 572.5). This dye was microinjected into the nucleoplasm of isolated Acetabularia nuclei (which are still surrounded by a thin layer of cytoplasm) and its subnuclear localization visualized by fluorescence microscopy. Striking differences, which seemed to be correlated with the developmental stage of the isolated nucleus, were observed. In nuclei isolated from cells at the stage of early cap stage formation, the dye was restricted to the nuclear envelope. In nuclei isolated from cells with intermediate or fully developed caps, there was increased nucleoplasmic staining, and the staining of the envelope was frequently diminished or abolished. In all nuclei, the dye remained within the nucleus after injection. Cytoplasmic staining was only observed when nuclei isolated from cells at the stage of early cap formation were incubated in a hyper- or hypo-tonic medium. Various ionophores, injected before the dye into the nucleoplasm, had no effect on the subsequent nuclear localization of DiOC₆(3), although they did rapidly induce nucleolar condensation in nuclei isolated from cells at the stage of early cap formation. The results suggested that the electrical properties of Acetabularia nuclear envelopes or inner nuclear membranes change during cell maturation. Furthermore, the retention of the dye in the nucleoplasm under isotonic conditions indicated that the nuclear pores were not open channels for molecules of this size.     

Ion channels in murine nuclei during early development and in fully differentiated adult cells

Summary The nuclear envelope functions as a selective barrier between nucleus and cytoplasm. During cycles of cell division the nuclear envelope repeatedly disassembles and re-associates. Presumably, each cycle re-establishes the functional and structural integrity of the nuclear envelope. After repeated rounds of cell division, as occurs during differentiation, the selectivity and configuration of the envelope may change. We compare the ionic conductance and the nuclear pore density in four types of murine nuclei: germinal vesicles in oocytes, pronuclei in zygotes, nuclei from two-cell blastomeres, and somatic cell nuclei from the liver. A large-conductance ion channel is present in all nuclear envelopes. Liver cell nuclei have a greater number of these channels than those from earlier developmental stages, and they also have a higher density of nuclear pores. In this article we hypothesize an association between the ion channels and the nuclear pores.     

膜片钳技术在动脉粥样硬化研究中的应用

膜片钳技术是一种先进的电生理技术,在生命科学研究中已得到了广泛的应用.最近几年已把它运用于研究动脉粥样硬化血管平滑肌细胞离子通道电生理特性的改变.研究发现血管平滑肌细胞的凋亡与K+通道活动增加有关,在动脉粥样硬化发生与发展过程中大电导型钙激活钾通道起着重要的功能作用.某些药物影响动脉粥样硬化血管平滑肌细胞离子通道而发挥作用.膜片钳技术给动脉粥样硬化发病机理研究带来了新的亮点.     

Properties of the mitochondrial peptide-sensitive cationic channel studied in planar bilayers and patches of giant liposomes.

A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed by the tip-dip method from proteoliposomes derived from mitochondrial membranes. It is blocked by peptide M, a 13 residue peptide having the properties of a mitochondrial signal sequence. To verify the reliability of the experimental approach, mitochondrial membranes from bovine adrenal cortex or porin-deficient mutant yeast were either fused to planar bilayers or incorporated in giant liposomes which were studied by patch clamp. Cationic channels were found with both techniques. They had the same conductance levels and voltage-dependence as those which have been described using the tip-dip method. Moreover, they were similarly blocked by peptide M. The voltage-dependence of block duration was analyzed in planar bilayer and tip-dip records. Results strengthen the idea that peptide M might cross the channel. Other mitochondrial channels were observed in planar bilayers and patch clamp of giant liposomes. Because they were never detected in tip-dip records, they are likely to be inactivated at the surface monolayer used to form the bilayer in this type of experiment.     

Localization of the cyclic ADP-ribose-dependent calcium signaling pathway in hepatocyte nucleus

CD38 is a type II transmembrane glycoprotein found on both hematopoietic and non-hematopoietic cells. It is known for its involvement in the metabolism of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate, two nucleotides with calcium mobilizing activity independent of inositol trisphosphate. It is generally believed that CD38 is an integral protein with ectoenzymatic activities found mainly on the plasma membrane. Here we show that enzymatically active CD38 is present intracellularly on the nuclear envelope of rat hepatocytes. CD38 isolated from rat liver nuclei possessed both ADP-ribosyl cyclase and NADase activity. Immunofluorescence studies on rat liver cryosections and isolated nuclei localized CD38 to the nuclear envelope of hepatocytes. Subcellular localization via immunoelectron microscopy showed that CD38 is located on the inner nuclear envelope. The isolated nuclei sequestered calcium in an ATP-dependent manner. cADPR elicited a rapid calcium release from the loaded nuclei, which was independent of inositol trisphosphate and was inhibited by 8-amino-cADPR, a specific antagonist of cADPR, and ryanodine. However, nicotinic acid adenine dinucleotide phosphate failed to elicit any calcium release from the nuclear calcium stores. The nuclear localization of CD38 shown in this study suggests a novel role of CD38 in intracellular calcium signaling for non-hematopoietic cells.     

Developmental regulation of a novel outwardly rectifying mechanosensitive anion channel in Caenorhabditis elegans

The nematode Caenorhabditis elegans offers unique experimental advantages for defining the molecular basis of anion channel function and regulation. However, the relative inaccessibility of somatic cells in adult animals greatly limits direct electrophysiological studies of channel activity. We developed methods to routinely isolate and patch clamp C. elegans embryo cells and oocytes and to culture and patch clamp neurons and muscle cells. Dissociated embryonic cells express a robust outwardly rectifying anion current that is activated by membrane stretch and depolarization. This current, termed I(Cl,mec), is inhibited by anion and mechanosensitive channel inhibitors. I(Cl,mec) has broad anion selectivity and the channel has a unitary conductance of 5-7 picosiemens. I(Cl,mec) is not detectable in whole-cell or isolated patch recordings from oocytes, cultured muscle cells, and cultured neurons but is expressed in single cell and later embryos. Channel density is high, and the current is observed in >80% of membrane patches. Macroscopic currents of 40-120 pA at +100 mV are typically observed in inside-out membrane patches formed using low resistance patch pipettes. Isolated membrane patches of early embryonic cells therefore contain 60-200 I(Cl,mec) channels. The apparent activation of I(Cl,mec) shortly after fertilization and its down-regulation in terminally differentiated cells suggests that the channel may play important roles in embryogenesis and/or cytokinesis.     

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.     

Fusion of native or reconstituted membranes to liposomes, optimized for single channel recording.

We here describe a protocol for fusing vesicles into large structures suitable for patch clamp recording. The method may be used with native membrane vesicles or with liposomes containing reconstituted/purified ion channels. The resulting unilamellar membranes exhibit high channel surface abundance, yielding multiple channels in the average excised patch. The procedure has been used to record voltage-sensitive Na channels from three native membrane preparations (eel electroplax, rat skeletal muscle, squid optic nerve), and from reconstituted protein purified from eel electroplax. Channels treated with batrachotoxin (BTX) displayed characteristic activation voltage dependence, conductances, selectivity, and sensitivity to saxitoxin (STX).     

Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.