Excised-patch

Our own laboratory obtained different results. Not only were we unable to see a clear cut correlation between the incidence of ICOR channels in cell attached patches and the degree of hormonal stimulation [57], we were also unable to reproduce the activation studies in excised patches. In our hands, the activation of ICOR channels occurred simply by the excision, and this was equally true for the normal as for the CF cells [57]. We did note, however, that the other laboratories worked at room temperature whereas we always work at 37°C. Welsh s laboratory has shown meanwhile that excision activation of ICOR Cl -channels is a temperature-dependent process [113]. At low temperature, excision activation is largely delayed [113] but it is immediate in our experiments at 37°C [57]. We concluded that the activation of the ICOR channels has probably little to do with phosphorylation but is rather due to the fact that the excised patch faces a new environment on the cytosolic side [57,72],... 

Or on excised patches of membrane, in which case the conditions on both sides of the membrane can be controlled. 

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.

Frog poison. Batrachotoxin (BTX) is a steroidal alkaloid from the skin of Phyllobates terribilis, a poisonous Colombian frog (source of the poison used on blowgun darts). In the presence of BTX, sodium channels in an excised patch stay persistently open when the membrane is depolarized. They close when the membrane is repolarized. Which transition is blocked by BTX ... 

The patch electrode voltage-clamp is the method of choice to study V, alterations (Hamill et al., 1981). However, the high resistance seals required for these studies are not readily formed on mammalian sperm due both to geometric factors as well as the low compliance of the plasma membrane (Arnoult et al., 1996b). To date, recordings of whole cell currents, as is essential for monitoring of V, have not been reported in sperm, despite the heroic efforts exerted to obtain excised patches from these cells (Espinosa et al., 1998). As an alternative, has been ex-... 

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. 

After performing the cell-attached patch, there is the possibility to obtain cell-free or excised-patches, to study single-channel currents on a... 

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. 

The mechanisms by which hypoxia regulates BK and other 02-sensitive K channels in neonatal chromaffin cells are not completely imderstood. These mechanisms are also relevant to neonatal rat CB type 1 cells (42) and neocortical neurons (43) where hypoxic regulation of BK channels did not occiu in excised patches, presumably owing to loss of cytoplasmic factors. This view was recently challenged in adult rat type 1 cells, where hypoxic regulation of BK channels was observed in inside-out patches and was proposed to occur by a membrane-delimited mechanism (44). While the reason for this discrepancy is imknown and may be age-related, the possibility that intracellular organelles (e.g., mitochondria) might remain attached to membrane patches after excision cannot be excluded (45). 

Riesco-Fagundo AM, Perez-Garcia MT, Gonzalez C, Lopez-Lopez JR. O2 modulates large-conductance Ca -dependent K+ channels of rat chemoreceptor cells hy a membrane-restricted and CO-sensitive mechanism. Circ Res 2001 89 430-436. Rustenheck I, Dickel C, Herrmann C, Giimmsmann T. Mitochondria present in excised patches from pancreatic B-cells may form microcompartments with ATP-dependent potassium channels. Biosci Rep 1999 19 89-98. 

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