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.     

A novel voltage clamp technique for mapping ionic currents from cultured skeletal myotubes.

The biophysical properties and cellular distribution of ion channels largely determine the input/output relationships of electrically excitable cells. A variety of patch pipette voltage clamp techniques are available to characterize ionic currents. However, when used by themselves, such techniques are not well suited to the task of mapping low-density channel distributions. We describe here a new voltage clamp method (the whole cell loose patch (WCLP) method) that combines whole-cell recording through a tight-seal pipette with focal extracellular stimulation through a loose-seal pipette. By moving the stimulation pipette across the cell surface and using a stationary whole-cell pipette to record the evoked patch currents, this method should be suitable for mapping channel distributions, even on large cells possessing low channel densities. When we applied this method to the study of currents in cultured chick myotubes, we found that the cell cable properties and the series resistance of the recording pipette caused significant filtering of the membrane currents, and that the filter characteristics depended in part upon the distance between the stimulating and recording pipettes. We describe here how we determined the filter impulse response for each loose-seal pipette placement and subsequently recovered accurate estimates of patch membrane current through deconvolution.     

The actions of intermediate and long-chain n-alkanols on unitary NMDA currents in hippocampal neurons.

The actions of the n-alkanols butanol, pentanol, and octanol on unitary currents passing through N-methyl-D-aspartate (NMDA) ion channels have been studied in cultured CA1 hippocampal neurons. The cell-attached patch clamp method, with L-homocysteic acid included in the patch pipette, was used to record single channel NMDA currents at the cell resting potential or for hyperpolarizing patch potentials. With the n-alkanols added to the bath solution, the mean open times for the NMDA channel were diminished and the channel conductance was unchanged. A decrease in mean open time to about 70% of control value was found with butanol (3 mM), pentanol (1 mM), and octanol (0.02 mM). In addition the n-alkanols had small effects to decrease the frequency of channel openings and to increase the amplitude of the unitary currents. The effects of the alcohols on intracellular calcium levels, during NMDA applications, were also measured using the fluorescent dye FURA II.     

Multiplicity of calcium permeabilities

In this review, we deal with recent progress on calcium currents in neuronal membrane. Most of the results discussed have come from the use of the patch clamp technique which allows one to record currents of small amplitude (a few tens of pA in the whole-cell configuration or less than the pA with excised patches), and to impose a calcium gradient across the membrane. This allows access to the elementary currents and a pharmacology of the Ca channels is developed.     

Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes.

To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive (MS) channel in Xenopus oocytes, currents were recorded from on-cell and excised patches of membrane with the patch clamp technique and from intact oocytes with the two-electrode voltage clamp technique. MS channels could be activated by stretch in inside-out, on-cell, and outside-out patch configurations, using pipettes formed of either borosilicate or soft glass. In inside-out patches formed with borosilicate glass pipettes, depolarizing voltage steps activated MS channels in a cooperative manner after delays of seconds. This voltage-dependent activation was not observed for outside-out patches. Voltage-dependent activation was also not observed when the borosilicate pipettes were either replaced with soft glass pipettes or coated with soft glass. When depolarizing voltage steps were applied to the whole oocyte with a two-electrode voltage clamp, currents that could be attributed to MS channels were not observed. Yet the same depolarizing steps activated MS channels in on-cell patches formed with borosilicate pipettes on the same oocyte. These observations suggest that the delayed cooperative activation of MS channels by depolarization is not an intrinsic property of the channels, but requires interaction between the membrane and patch pipette.     

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.     

Receptor-activated single channels in intact human platelets.

We report "cell-attached" patch clamp studies of intact human platelets which show receptor-activated single channels. Inclusion of ADP in the patch pipette, but not in the bath, resulted in the appearance of inward currents indicative of single channels tightly coupled to the ADP receptors. The channels had a slope conductance of 11 picosiemens at the resting potential. Removal of 1 mM Ca2+ or replacement of chloride by gluconate in the pipette filling solution had little effect on the slope conductance at the resting potential or on the estimated reversed potential. With isotonic BaCl2 in the pipette, ADP evoked single channel currents with a slope conductance of 10 picosiemens. Thus these channels appear to be permeable to monovalent and divalent cations and selective for cations over anions. Addition of 5 mM Ni2+ (which blocks ADP-evoked rapid calcium entry in fura-2-loaded platelets) to the pipette solution blocked ADP-evoked channel activity. These channels may therefore provide an important mechanism for ADP to activate human platelets within a small fraction of a second.     

新鲜分离小鼠胃ICC样细胞的鉴定及其电生理学特性

将免疫荧光及传统全细胞膜片钳技术应用于新鲜分离的小鼠胃Cajal 间质细胞样细胞上,探讨了小鼠胃Cajal 间质细胞样细胞形态和电生理学特性。经胶原酶消化得到的Cajal间质细胞样细胞胞体呈短梭形,且自胞体发出多个较短的毛刺状突起。免疫细胞化学结果表明,Cajal 间质细胞样细胞胞体和突起酪氨酸激酶受体c-kit表达呈阳性。在传统全细胞记录模式、膜电位钳制在-60 mV 的条件下,可以记录到自发、节律性内向电流,即起搏电流。钙调蛋白抑制剂W-7 (50µmol/L）明显增强了起搏电流幅度并引发明显的内向钳制电流。当电极内液中EGTA 的浓度由0.1 mmol/L增加到10 mmol/L时,也明显增强了起搏电流幅度并引发明显的内向钳制电流。实验结果提示,新鲜分离的小鼠胃Cajal 间质细胞样细胞可以产生自发、自律性内向电流,而这种电流对胞内低钙或钙调蛋白抑制剂敏感。这种具有自发性电活动的Cajal 间质细胞样细胞可能就是胃Cajal 间质细胞。     

Epidermal growth factor activates calcium-permeable channels in A 431 cells

The patch clamp technique in a cell-attached configuration was used to search for calcium-permeable channels in human carcinoma A 431 cells. Unitary inward currents were recorded with 100 mM CaCl2 in a pipette, with the mean slope conductance of 2.8 pS and a reversal potential (obtained by extrapolation) of +25.5 mV. Application of epidermal growth factor (EGF) into the extracellular solution produced a transient increase in the probability of these channels being open. The effect develops with delay of about 20 s and lasts thereafter for 36 s (mean values). We propose that these channels mediate an EGF-induced increase in the concentration of cytosolic free calcium.     

Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation

TRPM2 is a member of the melastatin-related TRP (transient receptor potential) subfamily. It is expressed in brain and lymphocytes and forms a cation channel that is activated by intracellular ADP-ribose and associated with cell death. In this study we investigated the calcium dependence of human TRPM2 expressed under a tetracycline-dependent promoter in HEK-293 cells. TRPM2 expression was associated with enhanced hydrogen peroxide-evoked intracellular calcium signals. In whole-cell patch clamp recordings, switching from barium- to calcium-containing extracellular solution markedly activated TRPM2 as long as ADP-ribose was in the patch pipette and exogenous intracellular calcium buffering was minimal. We suggest this effect reveals a critical dependence of TRPM2 channel activity on intracellular calcium. In the absence of extracellular calcium we observed concentration-dependent activation of TRPM2 channels by calcium delivered from the patch pipette (EC(50) 340 nM, slope 4.9); the maximum effect was at least as large as that evoked by extracellular calcium. Intracellular dialysis of cells with high concentrations of EGTA or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly reduced the amplitude of the extracellular calcium response, and the residual response was abolished by a mixture of high and low affinity calcium buffers. TRPM2 channel currents in inside-out patches showed a strong requirement for Ca(2+) at the intracellular face of the membrane. We suggest that calcium entering via TRPM2 proteins acts at an intracellular calcium sensor closely associated with the channel, providing essential positive feedback for channel activation.     

蛇床子素对L-和N-型钙通道的影响

目的:比较蛇床子素对不同钙通道亚型的作用差异方法:首先在tsA201细胞上瞬时转染Cav1.2,Cav1.3,Cav2.2e[37a],和Cav2.2e[37b]通道,然后采用全细胞膜片钳技术,记录tsA201细胞上的钙电流,并观察蛇床子素对各种钙通道亚型的影响结果:蛇床子素可以浓度依赖性抑制Cav1.2和Cav1.3电流,抑制的半有效浓度分别为162.1μmol·L^-1和56.2μmol·L^-1。此外,蛇床子素对Cav2.2通道也有一定的抑制作用,在300μmol·L^-1的浓度下,抑制38%的Cav2.2e[37a]电流和61%的Cav2.2e[37b]电流蛇床子素对钙电流的抑制是快速可逆的蛇床子素在各个测试电位水平均能抑制上述四种钙通道电流,但不改变电流的激活阈值和最大峰值电流的激活电压。结论:蛇床子素以浓度依赖的方式抑制多种钙通道亚型并表现出不同的亲和力     

CFTR Channels Expressed in CHO Cells Do Not Have Detectable ATP Conductance

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-dependent chloride channel which may have additional functions. Recent reports that CFTR mediates substantial electrodiffusion of ATP from epithelial cells have led to the proposal that CFTR regulates other ion channels through an autocrine mechanism involving ATP. The aim of this study was to determine the ATP conductance of wild-type CFTR channels stably expressed in Chinese hamster ovary cells using patch clamp techniques. In the cell-attached configuration with 100 mm Mg · ATP or Tris · ATP solution in the pipette and 140 mm NaCl in the bath, exposing cells to forskolin caused the activation of a low-conductance channel having kinetics resembling those of CFTR. Single channel currents were negative at the resting membrane potential (V _m ), consistent with net diffusion of Cl from the cell into the pipette. The transitions decreased in amplitude, but did not reverse direction, as V _m was clamped at increasingly positive potentials to enhance the driving force for inward ATP flow (>+80 mV). In excised patches, single channel currents did not reverse under essentially biionic conditions (Cl_in/ATP_out or ATP_in/Cl_out), although PKA-activated currents were clearly visible in the same patches at voltages where they would be carried by chloride ions. Moreover, with NaCl solution in the bath and a mixture of ATP and Cl in the pipette, the single channel I/V curve reversed at the predicted equilibrium potential for chloride. CFTR channel currents disappeared when patches were exposed to symmetrical ATP solutions and were restored by reexposure to Cl solution. Finally, in the whole-cell configuration with NaCl in the bath and 100 mm MgATP or TrisATP in the pipette, cAMP-stimulated cells had time-independent, outwardly rectifying currents consistent with CFTR selectivity for external Cl over internal ATP. Whole-cell currents reversed near V _m =−55 mV under these conditions, however the whole cell resistance measured at −100 mV was comparable to that of the gigaohm seal between the plasma membrane and glass pipette (7 GΩ). We conclude that CFTR does not mediate detectable electrodiffusion of ATP. Received: 8 November 1995/Revised: 23 January 1996     

Ginsenosides regulate ligand-gated ion channels from the outside

Treatment with ginsenosides, the major active ingredients of Panax ginseng, produces a variety of physiological effects on the central and peripheral nervous systems. Ginsenosides inhibit various types of ligand-gated ion channel but it is not clear whether they act from within or outside the cell since they are somewhat membrane-permeable. In the present study, we used the Xenopus oocyte gene expression system to determine from which side of the cell membrane the ginsenoside Rg3 (Rg3), and M4, a ginsenoside metabolite, act to regulate ligand-gated ion channel activity. Ligand-gated ion currents were measured using the two-electrode voltage clamp technique. Rg3 and M4 inhibited 5-HT3A and a3b4 nACh receptor-mediated ion currents when present outside of the cell but not when injected intracellularly. We also examined the effect of these agents on oocytes expressing the gustatory cGMP-gated ion channel, which is known to have a cGMP binding site on the intracellular side of the plasma membrane and is only activated by cytosolic cGMP. Rg3 inhibited cGMP-gated ion currents when applied extracellularly or to an outside-out patch clamp, but not when injected into the cytosol or when using an excised inside-out patch clamp. These results indicate that Rg3 and M4 regulate ligand-gated ion channel activity from the extracellular side.     

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 microm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and "patchability" of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66+/-0.07 microF/cm(2)) and conductance (23-44 microS/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

On the interaction of neomycin with the slow vacuolar channel of Arabidopsis thaliana

This study investigates the interaction of the aminoglycoside antibiotic neomycin with the slow vacuolar (SV) channel in vacuoles from Arabidopsis thaliana mesophyll cells. Patch-clamp experiments in the excised patch configuration revealed a complex pattern of neomycin effects on the channel: applied at concentrations in the submicromolar to millimolar range neomycin (a) blocked macroscopic SV currents in a voltage- and concentration-dependent manner, (b) slowed down activation and deactivation kinetics of the channel, and most interestingly, (c) at concentrations above 10 muM, neomycin shifted the SV activation threshold towards negative membrane potentials, causing a two-phasic activation at high concentrations. Single channel experiments showed that neomycin causes these macroscopic effects by combining a decrease of the single channel conductance with a concomitant increase of the channel's open probability. Our results clearly demonstrate that the SV channel can be activated at physiologically relevant tonoplast potentials in the presence of an organic effector molecule. We therefore propose the existence of a cellular equivalent regulating the activity of the SV channel in vivo.     

组胺拮抗剂对小鼠ATP-敏感性钾离子通道的影响

目的观察比较3种组胺拮抗剂对缺血性心肌细胞的ATP-敏感性钾离子通道中的影响。方法利用急性酶解法分离小鼠心室肌细胞。结果组胺拮抗剂pyrilamine、chlorpheniramine及diphenhydramine均可抑制ATP-敏感性钾离子通道的活性,抑制程度为pyrilamine〉chlorpheniramine〉diphenhydramine。组胺对KATP通道活性无影响。结论第一代的组胺拮抗剂（pyrilamine、chlorpheniramine及diphenhydramine）对KATP通道活性有抑制作用,其抑制作用与膜上H1受体无关。     

Cyclic adenosine monophosphate regulates calcium channels in the plasma membrane of Arabidopsis leaf guard and mesophyll cells

The effect of cAMP on Ca(2+)-permeable channels from Arabidopsis thaliana leaf guard cell and mesophyll cell protoplasts was studied using the patch clamp technique. In the whole cell configuration, dibutyryl cAMP was found to increase a hyperpolarization-activated Ba(2+) conductance (I(Ba)). The increase of I(Ba) was blocked by the addition of GdCl(3). In excised outside-out patches, the addition of dibutyryl cAMP consistently activated a channel with particularly fast gating kinetics. Current/voltage analyses indicated a single channel conductance of approximately 13 picosiemens. In patches where we measured some channel activity prior to cAMP application, the data suggest that cAMP enhances channel activity without affecting the single channel conductance. The cAMP activation of these channels was reversible upon washout. The results obtained with excised patches indicate that the cAMP-activated I(Ba) seen in the whole cell configuration could be explained by a direct effect of cAMP on the Ca(2+) channel itself or a close entity to the channel. This work represents the first demonstration using patch clamp analysis of the presence in plant cell membranes of an ion channel directly activated by cAMP.     

A whole-cell patch clamp technique which minimizes cell dialysis

A variant of the whole-cell patch clamp technique is described which allows measurement of whole-cell ionic currents in small cells while minimizing cell dialysis with the pipette solution. The technique involves the application of negative pressure to the inside of small (less than 1 micron) tip diameter pipettes placed on the cell surface to achieve high resistance seals and membrane rupture. The technique has been used successfully in a variety of different types of cells to study membrane currents carried by Ca and K, currents generated by exchange carriers as well as electrical coupling between cells. Overall, the technique seems well suited for the study of ionic currents in small cells, and provides an alternative to conventional patch clamping techniques which necessitate intracellular dialysis.     

A whole-cell patch clamp technique which minimizes cell dialysis

Summary A variant of the whole-cell patch clamp technique is described which allows measurement of whole-cell ionic currents in small cells while minimizing cell dialysis with the pipette solution. The technique involves the application of negative pressure to the inside of small (< 1 µm) tip diameter pipettes placed on the cell surface to achieve high resistance seals and membrane rupture. The technique has been used successfully in a variety of different types of cells to study membrane currents carried by Ca and K, currents generated by exchange carriers as well as electrical coupling between cells. Overall, the technique seems well suited for the study of ionic currents in small cells, and provides an alternative to conventional patch clamping techniques which necessitate intracellular dialysis.     

A comparison of calcium-activated potassium channel currents in cell-attached and excised patches

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.