Whole-cell patch-clamp techniques

We have launched an extensive study of calcium channels in connection with insecticidal action. First, we had to characterize the normal physiological properties of calcium channels in neuroblastoma cells as they were largely unknown. Whole cell patch clamp technique as combined with internal perfusion of neuroblastoma cells (N1E-115) (51) has proved highly successful as described below. 

The electrophysiological technique used to measure changes in membrane capacitance is the patch clamp [5,6] in the whole-cell recording mode, where the plasma membrane patch in the pipet is ruptured. In another configuration of the patch clamp, the plasma membrane patch is maintained intact. In this case, small currents due to the opening of individual channels can be measured in the membrane patch. The whole-cell patch clamp... 

INTESTINE Characterization of a membrane potassium ion conductance in intestinal secretory cells using whole cell patch-clamp and calcium-sensitive dye techniques, 192, 309 isolation of intestinal epithelial cells and evaluation of transport functions, 192, 324 isolation of enterocyte membranes, 192, 341 established intestinal cell lines as model systems for electrolyte transport studies, 192, 354 sodium chloride transport pathways in intestinal membrane vesicles, 192, 389 advantages and limitations of vesicles for the characterization and the kinetic analysis of transport systems, 192, 409 isolation and reconstitution of the sodium-de-pendent glucose transporter, 192, 438 calcium transport by intestinal epithelial cell basolateral membrane, 192, 448 electrical measurements in large intestine (including cecum, colon, rectum), 192, 459... 

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 patch clamp technique suddenly became the workhorse of modern electrophysiology, and the whole-cell configuration became one of the most crucial and popular techniques for the biophysical and pharmacological study of ion channels. 

By application of the whole-cell mode of the patch clamp technique to freshly isolated cells of convoluted proximal tubules, the sodium-alanine cotransport system could be investigated in detail (Hoyer and Gogelein 1991). 

Current variants of this technique make possible the application of solution on the exterior and interior of whole cells and on the membrane patches torn from the cell (outside-out or inside-out)—every thinkable configuration of solution and ion channel orientation craved by the ion channel researcher. Usually, primary cultured cells or cell lines are preferred as they reveal a relatively clean surface membrane (44) and require no enzymatic treatment that damages the plasmamembrane. The patch clamp technique is now the gold standard measurement for characterizing and studying ion channels and is one of the most important methods applied to physiology. 

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. 

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