Excised membrane patch

In respiratory epithelial (RE) cells the Cl -conductance was attributed to the ICOR channel. In fact, it was reported by Frizzell et al. and Welsh s laboratories that catecholamines increased the incidence of ICOR channels in cell attached patches of normal RE cells but failed to do so in CF cells [110,111], Later both laboratories presented data on excised membrane patches of RE cells in which the protein kinase A which was added to the cytosolic side produced ICOR channel activity in the normal cells but not in the CF tissues [19,20]. This finding was reproduced by Guggino and coworkers [22] for RE cells and by others for lymphocytes [46]. Protein kinase C at physiological Ca -activities had a comparable effect in normal cells but also failed to function in CF cells [22,112]. 

In our patch clamp studies in excised membrane patches in which we attempted to characterize the Cl we have noted that Cl did not only inhibit the probability of the ICOR channel being open but we also found that the input conductance of the patch was reduced at the same time and with the same time course [72]. We have followed up on this observation and we were able to show that this reduction in input conductance is caused by an inhibition of small ( lOpS) Cl -channels. Hence, we postulate that the same patches containing ICOR channels also contain small (unresolved, cf. section 2.4) Cl -channels which are inhibited reversibly by CL It cannot be excluded at this stage that these small Cl -channels are responsible for the defect in CF. 

Kawahara M, Arispe N, Kuroda Y, Rojas E. Alzheimer s disease amyloid beta-protein forms Zn(2+)-sensitive, cation-selective channels across excised membrane patches from hypothalamic neurons. Biophys J. 1997 73(1) 67-75. 

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. 

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 4. Effects of 60 pM (+)-trans tetramethrin on single sodium channels in an inside-out membrane patch excised from a neuroblastoma cell (N1E-115 line). A, sample records of sodium channel currents (inward deflections) associated with step depolarizations from -90 mV to -50 mV. B, as in A, but after application of tetramethrin to the internal surface of the membrane. C, current amplitude histogram in the control. D, as in C, but after application of tetramethrin. (Reproduced with permission from ref. 31. Copyright 1983 Elsevier.) Continued on next page.

Figure 16.16 Data from Kca1-ipi-subunit knockout (KO) mouse urinary bladder smooth muscle cells Indicate that the Kca1-1 (BK) channels from pi-subunlt KO mice have a reduced open probability compared to control (normal wild type) mouse. (A) Illustrates single channel recordings from excised Inside-out membrane patches (Section 16.5.1.3.) held at 40 and -i-40 mV In 10 gM free Ca + and symmetrical 60 gM K+ concentration. Arrows indicate the closed state of the channels. (B) illustrates BK channel open probability in control and pi-subunit KO animals at two different voltages (-40 and +40 mV). Data were obtained from n=9 11 cells of each animal group. Asterisks indicate statistically significant difference. Used with permission from Petkov et al., 2001a, J. Physiol. 537 443-452 - Copyright 2001 The Physiological Society.

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. 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.

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). 

Even beyond this, it should be clear that the large clamp voltage of say 100 mV may already lead to the inactivation of larger channels, and, still worse, the excision of the cell membrane itself may inactivate channels. It is not surprising then that the current literature gives a grossly distorted view of the world of Cl -channels. It is full of large and intermediate channels, but much less data are available on small channels. This analytical problem can be overcome by other patch clamp techniques. 

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.)...

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

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 ... 

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