The ultrastructure of patch-clamped membranes: a study using high voltage electron microscopy

We have developed techniques for studying patch-clamped membranes inside glass pipettes using high voltage electron microscopy (HVEM). To preserve the patch structure with the least possible distortion, we rapidly froze and freeze dried the pipette tip. The pipette is transparent for more than 50 microns from the tip. HVEM images of patches confirm light microscopy observations that the patch is not a bare bilayer, but a membrane-covered bleb of cytoplasm that may include organelles and cytoskeleton. The membrane that spans the pipette is commonly tens of micrometers from the tip of the pipette and occasionally as far as 100 microns. The structure of patches taken from a single cell type is variable but there are consistent differences between patches made from different cell types. With suction applied to the pipette before seal formation, we have seen in the light microscope vesicles swept from the plasmalemma up the pipette. These vesicles are visible in electron micrographs, particularly those made from chick cardiac muscle. Colloidal gold labeling of the patch permitted identification of lectin-binding sites and acetylcholine receptors. In young cultures of Xenopus myocytes, the receptors were diffuse. In 1-wk-old cultures, the receptors formed densely packed arrays. The patch pipette can serve, not only as a recording device, but as a tool for sampling discrete regions of the cell surface. Because the pipette has a constant path length for axial rotation, it is a unique specimen holder for microtomography. We have made preliminary tomographic reconstructions of a patch from Xenopus oocyte.     

Quantitative video microscopy of patch clamped membranes stress, strain, capacitance, and stretch channel activation.

Membrane patches from chick skeletal muscle were stretched by applying controlled suction or pressure to the pipette. From images of the patch, the patch dimensions (area and radius of curvature) were computed by nonlinear regression of the images to a geometric model. With no applied pressure, patch membranes are nearly planar and normal to the wall of the pipette. With increasing pressure gradients, the patch bulges, the radius of curvature decreases, and the area increases. The patch capacitance changes in exact proportion to the change in area at a rate of 0.7 microF/cm2. The increase in area is due to a flow of lipid (with perhaps small amounts of diffusible protein) along the walls of the pipette into the patch. The flow is reversible with a relaxation of the pressure gradient. The area elastic constant of the membrane is approximately 50 dyn/cm, insensitive to cytochalasin B and probably represents the elasticity of the underlying spectrin/dystrophin network. Simultaneous measurements of stretch activated (SA) ion channel activity in the patch showed that the sensitivity of channels from different patches, although different when calculated as a function of applied pressure, was the same when calculated as a function of tension. Because patch lipid is free to flow, and hence stress-free in the steady state, SA channels must be activated by tension in the cytoskeleton.     

Lipid-glass adhesion in giga-sealed patch-clamped membranes.

Adhesion between patch-clamped lipid membranes and glass micropipettes is measured by high contrast video imaging of the mechanical response to the application of suction pressure across the patch. The free patch of membrane reversibly alters both its contact angle and radius of curvature on pressure changes. The assumption that an adhesive force between the membrane and the pipette can sustain normal tension up to a maximum Ta at the edge of the free patch accounts for the observed mechanical responses. When the normal component of the pressure-induced membrane tension exceeds Ta membrane at the contact point between the free patch and the lipid-glass interface is pulled away from the pipette wall, resulting in a decreased radius of curvature for the patch and an increased contact angle. Measurements of the membrane radius of curvature as a function of the suction pressure and pipette radius determine line adhesion tensions Ta which range from 0.5 to 4.0 dyn/cm. Similar behavior of patch-clamped cell membranes implies similar adhesion mechanics.     

Cell membrane patches are supported by proteoglycans

A cell membrane patch isolated on a patch clamp pipette incorporates in addition to the phospholipid bilayer, an extracellular matrix and cytoskeletal components. The significance of the extracellular matrix for the patch formation was studied in aortic smooth muscle and cerebellar granule cells grow in the presence of an inhibitor of proteoglycan synthesis, -d -xyloside. The xyloside improved the seal success rate, and after patch excision membrane vesicles were formed instead of inside-out patches. When amphotericin B was included in the pipette solution, perforated outside-out vesicles were formed in 96% of cells. The findings suggest, that membrane patches are supported by the extracellular matrix or by structures that relate to this matrix.     

A look at membrane patches with a scanning force microscope.

We combined scanning force microscopy with patch-clamp techniques in the same experimental setup and obtained images of excised membrane patches spanning the tip of a glass pipette. These images indicate that cytoskeleton structures are still present in such membrane patches and form a strong connection between the membrane and the glass wall. This gives the membrane patch the appearance of a tent, stabilized by a scaffold of ropes. The lateral resolution of the images depends strongly on the observed structures and can reach values as low as 10 nm on the cytoskeleton elements of a (inside-out) patch. The observations suggest that measurements of membrane elasticity can be made, opening the way for further studies on mechanical properties of cell membranes.     

Currents recorded through small areas of squid axon membrane with an internal virtual ground voltage clamp.

A new voltage-clamp apparatus for the squid axon has been implemented to enable recording of currents through small areas of axon membrane. The performance of this clamp was tested by recording total sodium currents from perfused axons (I total) and sodium currents from small membrane patches (I patch), which were recorded from inside the axon with an L-shaped pipette. The I patch records, although four orders of magnitude smaller than I total, were stable and showed normal kinetics and voltage dependence, and appeared to reflect the activation of a small population of normal sodium channels. The size of the current recorded from the patch was mainly a function of the tip diameter of the L-shaped pipette and of the shunt resistance between inside the pipette and the axoplasm.     

Biophysics and Structure of the Patch and the Gigaseal

Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ components including ion channels. There is a 1-2 μm region of the seal below the patch where proteins are excluded and this may consist of extracted lipids that form the gigaseal. Patch domes often have complex geometries with inhomogeneous stresses due to the membrane-glass adhesion energy (E_a), cytoskeletal forces, and possible lipid subdomains. The resting tension in the patch dome ranges from 1-4 mN/m, a significant fraction of the lytic tension of a bilayer (∼10 mN/m). Thus, all patch experiments are conducted under substantial, and uneven, resting tension that may alter the kinetics of many channels. E_a seems dominated by van der Waals attraction overlaid with a normally repulsive Coulombic force. High ionic strength pipette saline increased E_a and, surprisingly, increased cytoskeletal rigidity in cell-attached patches. Low pH pipette saline also increased E_a and reduced the seal selectivity for cations, presumably by neutralizing the membrane surface charge. The seal is a negatively charged, cation selective, space with a resistance of ∼7 gigohm/μm in 100 mM KCl, and the high resistivity of the space may result from the presence of high viscosity glycoproteins. Patches creep up the pipette over time with voltage independent and voltage dependent components. Voltage-independent creep is expected from the capillary attraction of E_a and the flow of fresh lipids from the cell. Voltage-dependent creep seems to arise from electroosmosis in the seal. Neutralization of negative charges on the seal membrane with low pH decreased the creep rate and reversed the direction of creep at positive pipette potentials.     

Gated, ion-selective channels observed with patch pipettes in the absence of membranes: novel properties of a gigaseal.

Gigaohm seals made between patch pipettes and hydrophobic substrates have a finite conductance which are cation-selective and capable of producing quantized gating indistinguishable from the gating of biological ion channels. The selectivity sequence and streaming potentials of these seals suggests the existence of a pore of similar dimensions to the nicotinic acetylcholine channel. The ionic selectivity of these seals appears similar to the seal selectivity observed with membrane patches (Fischmeister, R., R. K. Ayer, and R. L. DeHann. 1986. Pfluegers Arch. 406:73-82) and the possibility of discrete gating within the seal region suggests caution when interpreting patch clamp data from unfamiliar preparations. The data suggests that the permeation pathway is the narrow space between the hydrophobic substrate and the pipette. Since this space has one hydrophobic wall, a hydrophilic channel lining may not be essential for channel permeation and gating.     

A localized zone of increased conductance progresses over the surface of the sea urchin egg during fertilization

Although activation of a sea urchin egg by sperm leads to three phases of membrane conductance increase in the egg, the mechanism by which the sperm causes these conductance changes is not known. We used the loose patch clamp technique to localize the conductance changes in voltage clamped eggs. A patch of the egg's membrane was isolated from the bath by pressing the loose patch clamp pipette against the egg surface. Sperm added to the bath attached to the surface of the egg in a region other than at the isolated membrane patch. During phase 1 of the activation current, no changes of the membrane conductance were detected. At the time of, and subsequent to the onset of phase 2, large currents recorded between the interior of the patch pipette and the bath were attributed to changes of the seal resistance between the surface of the egg and the pipette. A local change of membrane conductance was observed during phase 2 despite the changes of seal resistance. During phase 2, the large amplitude and short duration of the local membrane conductance increase relative to the membrane, conductance increase for the whole egg during phase 2 indicated that the conductance increase occurred over the entire surface of the egg, but not simultaneously. The time when the peak conductance for the membrane patch occurred, relative to the time of onset for phase 2 in the whole egg, depended on the distance, measured in a straight line, between the site of sperm attachment and the tip of the pipette. These data indicate that the localized conductance increase progressed over the surface of the egg from the site of sperm attachment to the opposite pole of the egg. It is proposed that the local conductance increase, the cortical reaction, and the change of seal resistance are all evoked by a common cytoplasmic message that progresses throughout the cytoplasm of the egg from the site of sperm attachment to the opposite pole of the egg.     

Exocytosis of single chromaffin granules in cell-free inside-out membrane patches

In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.     

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.     

Lipid bilayer mechanics in a pipette with glass-bilayer adhesion

Electrophysiology is a central tool for measuring how different driving forces (e.g., ligand concentration, transmembrane voltage, or lateral tension) cause a channel protein to gate. Upon formation of the high resistance seal between a lipid bilayer and a glass pipette, the so-called “giga-seal”, channel activity can be recorded electrically. In this article, we explore the implications of giga-seal formation on the mechanical state of a lipid bilayer patch. We use a mechanical model for the free energy of bilayer geometry in the presence of glass-bilayer adhesion to draw three potentially important conclusions. First, we use our adhesion model to derive an explicit relationship between applied pressure and patch shape that is consistent with the Laplace-Young Law, giving an alternative method of calculating patch tension under pressure. With knowledge of the adhesion constant, which we find to be in the range ∼0.4–4 mN/m, and the pipette size, one can precisely calculate the patch tension as a function of pressure, without the difficultly of obtaining an optical measurement of the bilayer radius of curvature. Second, we use data from previous electrophysiological experiments to show that over a wide range of lipids, the resting tension on a electrophysiological patch is highly variable and can be 10–100 times higher than estimates of the tension in a typical cell membrane. This suggests that electrophysiological experiments may be systematically altering channel-gating characteristics and querying the channels under conditions that are not the same as their physiological counterparts. Third, we show that reversible adhesion leads to a predictable change in the population response of gating channels in a bilayer patch.     

A pressure-polishing set-up to fabricate patch pipettes that seal on virtually any membrane,yielding low access resistance and efficient intracellular perfusion

When performing whole-cell configuration recordings, it is important to minimize series resistance to reduce the time constant of charging the cell membrane capacitance and to reduce error in membrane potential control. To this end, an existing method was improved by widening the patch pipette shank through the calibrated combination of heat and air pressure. The heat was produced by passing current through a filament that was shaped appropriately to ensure a homogeneous heating of the pipette shank. Pressurized air was applied to the lumen of a pipette, pulled from a borosilicate glass microcap, via the pressure port of a modified commercial holder. The pipette reshaping was viewed on an LCD monitor connected to a contrast-intensified CCD camera and coupled to a modified bright-field stereomicroscope. By appropriately regulating the timing of air pressure and the application of heating, the pipette shank and, independently, the tip opening diameter were widened as desired. The methods illustrated here to fabricate and use the patch pipettes, using just one glass type, allowed the sealing of a wide variety of cell types isolated from different amphibian, reptilian, fish, and mammalian tissues as well as a variety of artificial membranes made with many different lipid mixtures. The access resistance yielded by pressure-polished pipettes was approximately one-fourth the size of the one attained with conventional pipettes; besides improving the electrical recordings, this minimized intracellular ion accumulation or depletion as well. Enlarged shank geometry allowed for fast intracellular perfusion as shown by fluorescence imaging, also via pulled quartz or plastic tubes, which could be inserted very close to the pipette tip.     

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.     

The breakdown of cell membranes by electrical and mechanical stress.

We attempted to determine whether mechanical tension and electrical stress couple to cause membrane breakdown in cells. Using cell-attached patches from HEK293 cells, we estimated the mechanically produced tension from the applied pressure and geometry of the patch. Voltage pulses of increasing amplitude were applied until we observed a sudden increase in conductance and capacitance. For pulses of 50 micros duration, breakdown required >0.5 V and was dependent on the tension. For pulses of 50-100 ms duration, breakdown required 0.2-0.4 V and was independent of tension. Apparently two physically different processes can lead to membrane breakdown. We could explain the response to the short, high-voltage pulses if breakdown occurred in the lipid bilayer. The critical electromechanical energy per unit area for breakdown by short pulses was approximately 4 dyne/cm, in agreement with earlier results on bilayers. Our data suggest that, at least in a patch, the bilayer may hold a significant fraction (approximately 40%) of the mean tension. To be compatible with the large, nonlytic area changes of patches, the bilayer appears to be pulled toward the pipette tip, perhaps by hydrophobic forces wetting membrane proteins bound to the glass. Although breakdown voltages for long pulses were in agreement with earlier work on algae, the mechanism(s) for this breakdown remain unclear.     

通过膜片钳玻璃微电极内插管进行胞内透析

本文介绍一种膜片钳微电极内插管进行胞内透析的方法。利用通用的微电极夹持器在其抽吸负压的侧管上方钻一斜孔直通夹持器中央管腔。插入微量移液管头拉制成的细管（外径约0.1mm）,后者与Ag-AgCl电极一起伸出夹持器口端。通过相连的注射器,可以很方便地进行电极内液置换及胞内药物透析,此法和二次钳压技术及国外介绍的微插管电极内液置换相比,更加简便易行,结果更可靠。     

Cellular dynamics of conjugation in the ciliate euplotes aediculatus. II. Cellular membranes

The formation and subsequent dissolution of a common bridge of cytoplasm between conjugating ciliated protozoan cells provides an excellent opportunity to follow the dynamics of the cellular membrane systems involved in this process. In particular, separation of conjugant partners offers the chance to observe, at a fixed site on the cell surface, how the ciliate surface complex of plasma and alveolar membranes (collectively termed the “pellicle”) is constructed. Consequently, cortical and cellular membranes of Euplotes aediculatus were studied by light and electron microscopy through the conjugation sequence. A conjugant fusion zone of shared cytoplasm elaborates between the partner cells within their respective oral fields (peristomes) to include microtubules, cytosol, and a concentrated endoplasmic reticulum (heavily stained by osmium impregnation techniques) that may also be continuous with cortical ER of each cell. Cortical membranes displacd by fusion are autolyzed in acid phosphatase-positive lysosomes in the fusion zone. As conjugants separate, expansion of the plasma membrane may occur through the fusion of vesicles with the plasma membrane, presumably at bare membrane, presumably at bare membrane patches near the fusion zone. The underlying cortical alveolar membranes and their plate-like contents are reconstructed beneath the plasma membrane, apparently by multiple fusions of dense-cored alveolar precursor vesicles (APVs). These precursor vesicles themselves appear to condense directly from the smooth ER present in the fusion zone. No Golgi apparatus was visible in the fusion zone cytoplasm, and no step of APV maturation that might involve the Golgi complex was noted.     

Cytoplasmic membrane systems involved in bacterium release into soybean nodule cells as studied with two Bradyrhizobium japonicum mutant strains

Two Bradyrhizobium japonicum, Tn5-induced, mutant strains, ML126 and ML150, were studied. Both induce host cell division to form normal-sized nodules that do not fix nitrogen and whose cells have very few bacteroids (Bar-). Early-infection (15 days post infection) cells have much endoplasmic reticulum (ER), numerous Golgi bodies, and large vacuoles that are probably secondary lysosomes. Later the cytoplasm of the host cells of both are dominated by hundreds of vesicles containing only finely fibrous material and that appear to originate by the degradation of the cell walls of the infection threads; they have been named "infection-thread wall degradation vesicles" (IWDV). Phosphotungstic acid-chromic acid (PACA) staining of thin sections shows that IWDV membranes and the plasma membranes of both the cells and infection threads usually stain quite intensely, while the membranes of other cell organelles do not. The membranes of the few symbiosomes present in the mutants also stain with PACA. This evidence suggests that largely the host-cell plasma membrane gives rise to both the vesicle and symbiosome membranes in these mutants. In cells induced by both mutants, ER appears to be deficient, a finding suggesting that an ER-synthesis signal is involved in the normal release process, that ER synthesis is prerequisite to a normal volume of release, and that insufficient ER can impair symbiosome formation. In the mutant-induced infections, normal lysosomes develop and engulf both symbiosomes and cytoplasmic vesicles, but the retardation of this activity is the probable cause of the cytoplasm becoming overloaded with vesicles.     

Seal-promoting solutions and pipette perfusion for patch clamping plant cells

Patch-clamp technology has greatly increased our knowledge of plant membrane transport. However, the success of patch clamping crucially relies on establishing a high resistance (GΩ) seal between the membrane and the patch-clamp pipette. This can prove problematic in many plant-cell preparations. It is therefore of great importance to develop protocols for protoplast isolation, maintenance and seal formation that improve seal rate. This study investigated whether the pH and the K⁺ and the Cl⁻ concentration of the pipette solution had an effect on the seal formation. High pH and absence of K⁺ significantly promoted membrane sealing, whereas the concentration of Cl⁻ had no effect. To reap the benefit of seal-promoting pipette solutions and yet retain the option to adjust this solution to experimental requirements, a pipette perfusion apparatus was implemented. The perfusion system was successfully applied in cell-attached patch, excised-patch and whole-cell configurations, using plasma membrane and tonoplast of three different species. The system enables complete solution exchange within minutes and is potentially of great benefit in the study of channel selectivity, the application of (cytoplasmic) channel blockers and the study of primary and secondary transport.     

High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation

Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 mus and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.