Configuration, cell-attached

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.)...

The most commonly used method is called the on-cell or cell-attached configuration, because ion channels can be recorded on an intact cell (Jackson 1993). This mode is well suited for investigation of ion channels, which are activated by hormonal stimulation and triggered by intracellular second messengers. 

Some of the first electrophysiological studies on the MTX-induced conductance were carried out in guinea pig cardiac muscle cells (Kobayashi, Ochi, and Ohizumi 1987) in which 0.09 tiM MTX produced a voltage-independent current carried by Ca and Ba but not Cd. In cell-attached patches, the single-chaimel conductance was 12 pS in the presence of 50 mM Ba. Further studies showed that 10 uM MTX induced a voltage-insensitive current carried mainly by Na, not choline, with a reversal potential close to 0 mV and a single chatmel conductance of 16 pS in cell-attached configuration. This current was independent of the presence of at both sides of the membrane patch (Nishio, Muramatsu, andYasumoto 1996). 

Figure 13.13. Patch-Clamp Modes. The patch-clamp technique for monitoring channel activity is highly versatile. A high-resistance seal (gigaseal) is formed between the pipette and a small patch of plasma membrane. This configuration is called cell attached. The breaking of the membrane patch by increased suction produces a low-resistance pathway between the pipette and interior of the cell. The activity of the channels in the entire plasma membrane can be monitored in this whole-cell mode. To prepare a membrane in the excised-patch mode, the pipette is pulled away from the cell. A piece of plasma membrane with its cytosolic side now facing the medium is monitored by the patch pipette.

Whole-cell mode—conventional. From the initial cell-attached configuration, additional suction is applied to rupture the cell membrane, thus providing access to the intracellular space of the cell. The larger opening at the tip of the patch electrode, compared with the sharp microelectrode, provides lower resistance and thus better electrical access to the cell. Because the volume of the patch electrode is much bigger than the cell, the soluble contents of the cell will slowly be replaced by the contents of the electrode, referred to as dialyzing ... 

Jiang et al. reported that Ginsenosides-Re, -Rgi, -Rg2 and-Rh had both calcium channel blockade and anti-ffee-radical actions, -Rf had blockade action on L type channel in cultured myocardiocytes of rats with the cell attached configuration of patch-clamp technique and electron spin resonance method[21]. Ginsenoside-Re could coordinately promote the colony formation and increase H-TdR incorporation of bone marrow cell[22]. 

Figure 1. Photomicrograph (4X) of polymer construct seeded with chondrocytes. Note the rounded configuration of the cells and the cell attachment to the polymer in multiple layers.

We felt that the physicochemical properties of the polymer might therefore be manipulated to alter the cell shape and thus the cell physiology. We next studied the effects of polymers of different physical and chemical configuration on cell attachment, viability and performance of differentiated function. The ability of the hepatocyte to maintain differentiated function was assessed by Ae rate of albumin secretion. We found that a suitable polymer was an uncoated 85 15 combination of polylactic/polyglycolic acid. This suggested to us that a... 

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... 

At the cell-attached configuration, the membrane potential under the patch pipette equals the resting potential of the cell minus the potential applied to the pipette. This means that when the pipette potential is 0, the membrane potential of the patch equals the cell resting potential, and a negative pipette potential will depolarize the membrane. When a negative current is observed, it can be interpreted as a cationic outward current, and the positive one is a cationic inward current. 

Since the cell integrity is maintained in the cell-attached configuration, all the biochemical mechanisms that may be linked to the channel function (as second messenger metabolism or phosphorilation mechanisms) are not disturbed. This provides the possibility to modify the metabolic conditions of the cell, by applying active substances to the bath solutions, and to observe the results on the channel activity (see for example ref. [25]). 

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.

Fig. 8. Whole-cell configuration. After obtaining the cell attached configuration (A), giving a negative pressure or potential pulse, the membrane under the pipette tip can be broken, and a direct access of the pipette to the cell interior is obtained (B). This process can be monitored by the change of the current recorded as response of small voltage pulse, where a significant increase of the capacitative component is observed. In whole-cell configuration, macroscopic currents can be observed, for example, as a response to membrane potential.

As with extracellular measurements, intracellular measurements can be made by combining electrophysiological and electrochemical techniques (120). For intracellular measurements, the initial patch electrochemical setup is in the cell attached configuration. However, after some time additional suction is applied to the patch pipette, the membrane is ruptured and the whole cell configuration is attained (Figure 17.1.15). Disruption of the membrane allows oxidizable intracellular content to diffuse to the UME where it is detected in either the amperometric or voltammetric mode. 

Figure 8. Schematics of the sanqile cell attached to the Au-coated coupling prism in this Kretschmaim configuration, and connected to a closed liquid handling system.

Seals are made by applying gentle suction to identified synaptic boutons visualized with Nomarski optics. Seal resistances of up to 30 G 2 form, and currents can be recorded in cell-attached, inside-out or whole-cell recording configurations (Martinez-Padron and Ferrus 1997). Records from enlarged, mutant type III boutons on muscle 12 (>2 im in diameter) showed an average input resistance of 8-10 G 2 and mean bouton capaci-... 

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