Cell-attached patch clamp technique

Using the cell-attached patch clamp technique on frog muscle fibers (79), one can observe only two conditions the open, conducting state of the receptor and a nonconducting state of unknown identity. The transitions behave according to stochastic principles the lifetimes of any particular condition are distributed exponentially. The open state has a mean duration that is the inverse of the rate of channel closing. Because channel open time depends only upon a conformational shift, agonist concentration does not influence the parameter. It is, however, influenced... 

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.

In the whole cell model, a gigaseal is formed as the pipette is attached to the cell, and then a more dynamic suction is applied, which causes the interior of the cell to be sucked into the pipette tip (Fig. 3a). This action allows current and conductance of the entire cell to be measured. Therefore, the whole cell model measures changes caused by many ion channels on the entire cell membrane. Additionally, the liquid content of the cell will mix and equilibrate with the solution in the pipette, which allows pharmacological agents to be administered into the cell. Of the patch clamp techniques, the whole cell method is the most common and can be used to determine how pharmacological agents affect the total conductance of neurons. 

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.

Figure 16.14 Original Kca1-1 (BK) single channel recordings and the inhibitory effect of iberiotoxin. Currents were recorded from a guinea pig urinary bladder smooth muscle cell using the whole cell (cell-attached), perforated patch-clamp technique at 0 mV holding potential (Section...

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]. 

Cell-attached (single-channel) voltage clamp Patch clamp technique Patch voltage clamp technique Voltage clamp... 

The structures of a number of channel proteins have been obtained by the X-ray diffraction techniques that will be described in greater detail in Chapter 12. Information about the flow of ions across channels and pumps is supphed by the patch clamp technique. One of many possible experimental arrangements is shown in Fig. 8.19. With mild suction, a patch of membrcme from a whole cell or a small section of a broken cell cm be attached tightly to the tip of a micropipette filled with an electrolyte solution and containing an electronic conductor, the patch electrode. A potential difference (the clamp ) is applied between the patch electrode and an intracellular electronic conductor in contact with the cytosol of the cell. If the membrane is permeable to ions at the applied potential difference, a current flows through the completed circuit. Using narrow micropipette tips with diameters of less than 1 pm, ion currents of a few picoamperes (1 pA = 10" A) have been measured across sections of membranes containing only one ion channel protein. 

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