Outside-out configuration

Selected entries from Methods in Enzymology [vol, page(s)] Technique and sample preparation, 207, 159 outside-out configuration, 207, 10 amplifiers, 238, 153, 310 scrape loading,... 

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. 

Outside-out configuration From the whole-cell configuration it is possible to achieve a new excised-patch. Withdrawing the patch pipette very slowly (Fig. 9A), a small membrane vesicle can be formed on the tip of the pipette (Fig. 9B). This is the outside-out configuration, where the outer surface of the membrane is exposed to the bath solution, and the internal surface of the patch to the pipette solution. In this configuration the pipette potential is equal to that of the membrane, and the recorded current convention is the same as that in whole-cell configuration. 

Fig. 9. Outside-out configuration. After obtaining the whole-cell configuration (A), the pipette is withdrawn, and an excised membrane forms an outside-out patch. When the outside-out patch is obtained, the capacity current observed in response to a small voltage pulse changes significantly, since a reduction of the capacity occurs (from whole-cell to a patch of membrane) (B). At this configuration, single-channel currents are recorded.

Singer and colleagues studied the effect of non-esterified arachidonate on potassium channels by dissolving the fatty acid in the bathing medium and perfusing it on the inside of the smooth muscle membrane (in the inside-out configuration) or on its outside (in the outside-out configuration). In either case, they found that application of micromolar concentrations of the fatty acid would cause the activation of a potassium-selective channel (Fig. 3.6). This effect was not mediated by the formation of... 

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.)...

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). 

Outside-Out Patch Clamp If the pipette is retracted while it is in the wholecell configuration, a membrane patch is produced that has its extracellular surface exposed. This arrangement is called the outside-out recording configuration. 

Inside-out, Outside-in Filtration in Hollow-Fiber Membranes Hollow-fiber membrane modules can be operated in two different flow modes— inside-out and outside-in —based on the direction of filtration flow. In the inside-out configuration, pressurized feed water flows through the bore of a hollow fiber, and permeate is collected on the outside of the membrane fibres. In the outside-in configuration, the pressurized feed stream flows from the outside of a hollow fiber, and permeate is collected inside the bore of the hollow fiber. 

Quench. Attempts have been made to model this nonisotherma1 process (32—35), but the complexity of the actual system makes quench design an art. Arrangements include straight-through, and outside-in and inside-out radial patterns (36). The optimum configuration depends on spinneret size, hole pattern, filament size, quench-chamber dimensions, take-up rate, and desired physical properties. Process continuity and final fiber properties are governed by the temperature profile and extension rate. 

Membrane systems consist of membrane elements or modules. For potable water treatment, NF and RO membrane modules are commonly fabricated in a spiral configuration. An important consideration of spiral elements is the design of the feed spacer, which promotes turbulence to reduce fouling. MF and UF membranes often use a hollow fiber geometry. This geometry does not require extensive pretreatment because the fibers can be periodically backwashed. Flow in these hollow fiber systems can be either from the inner lumen of the membrane fiber to the outside (inside-out flow) or from the outside to the inside of the fibers (outside-in flow). Tubular NF membranes are now just entering the marketplace. 

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