Patch pipette

Preliminary data, in which extracellular and intracellular glutathione status were altered in the absence of a ffee-radical-induced oxidant stress have demonstrated a similar effect on /Na/K- In these experiments the extracellular concentration of GSH was altered by varying the levels in the extracellular perfusate. In contrast, the intracellular GSH content was controlled by the inclusion of the required concentration of GSH in the patch pipette. After 5 min exposure to GSH there was an approximately 20% increase in /na/K at 0 mV. Conversely, in a separate group of cells, 5 min after the application of GSSG there was an approximately 15% decrease in /wa/K (Haddock, 1991). 

Fig. 21.11. (Opposite) (A) Representative vesicle-attached patch recordings from SENS, PYRR and LEVR isolates 30 pM levamisole in the patch-pipette, -50 mV patch potential. Note that the SENS isolate recording contains more openings than the other two patches and so will carry more current across the membrane. (B) Log10 plot of the proportion of open-time against the isolate type. Open circles represent individual receptor channels from different preparations at -50 mV with 30 pM levamisole as the agonist. Note that there is a wide spread of greater than tenfold difference between the maximum and minimum values observed for each of the isolates. There is overlap between values of the isolates but the mean (closed square) for SENS is greater than for LEVR and PYRR.

Fig. 21.14. Isolated inside-out patch from pharyngeal muscle with a patch potential of +50 mV and 800 pM glutamate present in the patch pipette shows the opening of two Glu-CI channels. The addition of 1 pM ivermectin to the bath and thus to the inside of the patch produces (in the same patch) opening of up to three Glu-CI channels simultaneously. Ivermectin increases the probability of opening of Glu-CI channels already opening in the presence of glutamate.

Kotlikoff We haven t done this. We first glued pipettes onto the end of cells. We then also found that just producing a seal with patch pipettes allows us to stretch cells quite nicely. This allows us to voltage clamp and record the currents at the same time, which we do in one of those experiments, demonstrating activation of a Ca2+-activated CD current. 

Wier What is the spatial resolution of your Ca2+ measurements In other words, are you picking up the edges of the wave before it actually arrives at your patch pipette, just reflecting limited spatial resolution ... 

McCarron We clamped the ATP via the patch pipette filling solution. 

Paul We had some pretty interesting data that without glycolytic substrates, even with ATP in a patch pipette it doesn t get where it is supposed to go (Lorenz Paul 1997). We can see really big changes with the glycolytic cocktails in the presence of 5 mM ATP. 

Sakmann B (2006) Patch pipettes are more useful than initially thought simultaneous pre- and postsynaptic recording from mammalian CNS synapses in vitro and in vivo. Pflugers Arch 453 249-259... 

Borosilicate glass capillaries for patch pipettes (World Precision Instruments, Sarasota, EL). 

Pull and fire polish patch pipettes when filled with buffer solution show a resistance of 3-5 MQ and a series resistance of 5-6 MQ. 

Another versatile mode is the cell-excised configuration (Hamill 1993). It is obtained by suddenly removing the patch-pipette from the cell, so that the membrane patch is pulled off the cell. This mode easily allows to expose the channel proteins to drugs by changing the bath solution. The single channel currents are recorded on a videotape and are analyzed off-line by a computer system. Various parameters are evaluated, such as the single channel conductance, open-and closed-times of the channel, and the open-state probability, which is the percentage of time the channel stays in its open state. 

Besides these modes, which enable the recording of single channel currents, it is also possible to measure the current flowing through the entire cell. This whole-cell mode is obtained by rupturing the membrane patch in the cell-attached mode (Hamill et al. 1981 Dietzel et al. 1993). This is achieved by applying suction to the interior of the patch-pipette. The whole-cell mode not only allows to record the electrical current, but also to measure the cell potential. Moreover, the cell interior is dialyzed by the electrolyte solution filled into the patch-pipette. 

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.

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. 

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