Inside-out membrane patches

Fig. 3. Summary of results defining conditions that lead to opening of muscarinic K channels. Panel A. Experiments by Soejima and Noma [111] showed that opening of K channels in an isolated membrane patch occurs only by stimulation of a receptor located in the same patch (top) but not by stimulation of receptors outside of the patch (bottom). B. Experiments of Pfaffinger et al. [142] showed that acetylcholine (ACh) cannot lead to opening of K+ channels unless GTP is supplied as a co-factor (top vs. bottom). C. Experiments by Kurachi et al. [144] and Yatani et al. [145] showed that addition of GTPyS to the inside face of inside-out membrane patches leads to agonist-independent opening of K channels. D. Activation of K+ channels, independent of receptor occupancy, occurs on addition of GTPyS-activated Gk (Gk ) or its resolved subunit (ak ), For details see Fig. 4 and Refs. 144 and 150.

Figure 4. Effects of 60 pM (+)-trans tetramethrin on single sodium channels in an inside-out membrane patch excised from a neuroblastoma cell (N1E-115 line). A, sample records of sodium channel currents (inward deflections) associated with step depolarizations from -90 mV to -50 mV. B, as in A, but after application of tetramethrin to the internal surface of the membrane. C, current amplitude histogram in the control. D, as in C, but after application of tetramethrin. (Reproduced with permission from ref. 31. Copyright 1983 Elsevier.) Continued on next page.

Figure 16.16 Data from Kca1-ipi-subunit knockout (KO) mouse urinary bladder smooth muscle cells Indicate that the Kca1-1 (BK) channels from pi-subunlt KO mice have a reduced open probability compared to control (normal wild type) mouse. (A) Illustrates single channel recordings from excised Inside-out membrane patches (Section 16.5.1.3.) held at 40 and -i-40 mV In 10 gM free Ca + and symmetrical 60 gM K+ concentration. Arrows indicate the closed state of the channels. (B) illustrates BK channel open probability in control and pi-subunit KO animals at two different voltages (-40 and +40 mV). Data were obtained from n=9 11 cells of each animal group. Asterisks indicate statistically significant difference. Used with permission from Petkov et al., 2001a, J. Physiol. 537 443-452 - Copyright 2001 The Physiological Society.

Figure 1 Schematic diagrams illustrating the patch-clamp technique. (A) Overall setup for isolating single ionic channels in an intact patch of cell membrane. P = patch pipet R = reference microelectrode I = intracellular microelectrode Vp = applied patch potential Em = membrane potential Vm = Em — Vp = potential across the patch A = patch-clamp amplifier. (From Ref. 90.) (B) Five different recording configurations, and procedures used to establish them, (i) Cell attached or intact patch (ii) open cell attached patch (iii) whole cell recording (iv) excised outside-out patch (v) excised inside-out patch. Key i = inside of the cell o = outside of the cell. (Adapted from Ref. 283.)...

Fig. 21.4. Vesicle formation and patch-clamp techniques used to record levamisole receptor channel currents from Ascaris muscle. (A) Muscle membrane vesicles bud-off from the bag membrane following a 10 min collagenase treatment and incubation for 1 h at 37°C in Ascaris saline. (B) Levamisole is applied to the outside surface of the membrane to activate receptor channels cell-attached patches are usually used but it is also possible to make inside-out and outside-out patch recordings.

Inside-out patch, where the electrode is quickly withdrawn from the cell when the gigaseal is formed, thus ripping the patch of membrane off the... 

Current variants of this technique make possible the application of solution on the exterior and interior of whole cells and on the membrane patches torn from the cell (outside-out or inside-out)—every thinkable configuration of solution and ion channel orientation craved by the ion channel researcher. Usually, primary cultured cells or cell lines are preferred as they reveal a relatively clean surface membrane (44) and require no enzymatic treatment that damages the plasmamembrane. The patch clamp technique is now the gold standard measurement for characterizing and studying ion channels and is one of the most important methods applied to physiology. 

In the outside-out model, the pipette is attached to the entire cell as in the whole cell model, followed by a sharp pull that causes the cell membrane to break and reseal with the pipette tip (Fig. 3b). With the extracellular region exposed, channel activity as a response to different external stimuli can be probed. This configuration is less common than the inside-out method. Using an outside-out method, single-channel opening activity has been recorded while various neurotransmitters were released. For example, this patch clamp method was used as a detector for capillary electrophoresis separations of GABA, glutamate, and NMDA (7). 

Horn Our results suggest that this probably won t be to do with the cytoskeleton. We see the same thing with a cell-attached patch and an inside—out patch. The cell-attached patch itself disrupts the cytoskeleton when you draw the membrane into the patch pipette. I think something else is going on when you pull it away. There is probably still some cytoskeleton in there, and I think the membrane is disrupted in inside-out patches. 

There are several variations of the patch-clamp technique (Figure 16.21). Ion channel currents can be recorded from a whole cell or from small membrane pieces called excised patches, which are sections that are physically excised and removed from the cell membrane. The excised patch can be either in inside-out or outside-out configuration. The different patch-clamp configurations are described later. 

The inside-out patch technique is to retract the pipette that is in the cell-attached configuration so that a small vesicle of membrane remains attached. By exposing the tip of the pipette to an external electrolyte, it is possible to control the medium to which the intracellular surface of the membrane is exposed. Alternatively, if the pipette is retracted while it is in the whole-cell configuration, the membrane ruptured ends at the tip of the... 

This is a true single-channel patch clamp mode allowing the measurement of singlechannel currents with the added benefit of making it possible to change the medium to which the intracellular surface of the membrane is exposed. Thus, the inside-out configuration is particularly valuable when studying the influence of intracellular molecules on ion channel function, like calcium or ATP. 

Fig. 1 Schematic diagram of the patch-damp technique [5] A heat-polished glass pipette (tip diameter in the order of l jim) is pressed against a clean membrane surface. Upon slight suction a high resistance seal (> 1 GO) forms between the tip of the pipette and the membrane. This high seal resistance and the small membrane area reduce the background noise in such a way, that it becomes possible to measure currents (< IpA) passing through a single ion channel protein. The mechanical stability of the seal between pipette and membrane allows the isolation of the membrane patch encircled by the pipette The withdrawal of the pipette in the attached configuration results in an inside-out patch.

Single-channel recordings of chloride channels in inside-out patches (the intrathylakoid membrane side faced the bath solution) under asymmetrical KCl concentrations 20 mM KCl,... 

Fig. 7. Inside-out patch. When the cell-attached configuration has been obtained (A), the pipette is pulled-up, and a patch of membrane is excised from the cell (B), forming the inside-out patch-clalmp configuration. In this configuration, single-channel currents are observed. When the excised membrane reseals, forming a small vesicle in the tip of the pipette, the channel current is distorted (C). In this case, the vesicle can be disrupted crossing the air-water interface (D), and the inside-out configuration is obtained.

We have studied the electrophysiological properties of outer mitochondria membranes incorporated in liposomes, using the patch-clamp technique. The proteoliposomes were prepared and enlarged in 150 mM KCl, 0.1 mM CaCh, and 20 mM HEPES, and the same solution was used in the recording pipette. Liposome-attached and inside-out patches were obtained. 

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