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Patch-Clamp Electrophysiology

Summary

All biological cells produce ionic gradients between their insides (cytoplasm) and outside. Many biological cells, including neurons, muscle cells, and immune cells utilize rapid changes in ionic conductances to create or react to biological signals. These conductances are controlled by ion channel proteins that traverse the cell membrane. Patch-clamping is a technique for measuring these ionic currents in either a small patch of a cell membrane or the entire cell. A great deal of information about how ion channels function can be obtained with this technique.

Electrical isolation of the patch or cell is obtained using a glass micropipette that adheres to the cell membrane, forming a very high-resistance seal. A wire electrode within the glass patch pipette detects the voltage within the pipette. A patch-clamp amplifier measures the voltage inside the pipette relative to an extracellular reference electrode (ground) and can amplify this voltage difference to levels that can be detected by common recording equipment. The patch-clamp amplifier can also be used in voltage-clamp mode. In this case, the voltage difference between the pipette electrode and the reference electrode is held at a fixed value by sending current (either positive or negative) into the pipette electrode. In voltage-clamp mode, the amplifier monitors the amount of current flowing through the pipette electrode and produces an output voltage signal that is directly proportional to the current.

Figure 1

Patch clamp electrophysiology can be performed in different configurations, depending on the experimental goals.  These include on-cell patch, excised inside-out patch, whole cell, and outside-out patch.  http://www.science-display.com/patchclamp.html.

Figure 2

Cell-attached patch

This is the simplest configuration, where a high-resistance (gigohm) seal between the patch pipette and a cell electrically isolates a small patch of membrane at the tip of the pipette. Currents through this small membrane patch can be monitored, but the driving voltage for this current is uncertain because the transmembrane voltage is between the inside of the pipette and the inside the cell (the other side of the patch) which may differ from that of the extracellular reference electrode.

Excised inside-out patch

After obtaining an on-cell seal, the membrane patch can be torn from the cell by withdrawing the pipette away from the cell, which adheres to the substrate (usually a tissue-culture dish or coverslip). This excised membrane patch is oriented with the inner cell membrane side facing outward. This configuration allows currents through the small membrane patch to be measured with a known driving voltage. Another difference between on-cell experiments and excised inside-out patch experiments is that intracellular (cytoplasmic) factors that may affect ion channel activity are lost after patch excision.

Whole-cell

After obtaining an on-cell seal, the patch at the tip of the membrane can be ruptured to create an electrical pathway between the inside of the patch pipette and the inside of the cell. This allows measurement of larger currents passing across the entire cell membrane surface. If these currents are too large, a voltage change across the high-resistance tip of the patch pipette develops, which alters the driving voltage. Patch-clamp amplifiers include additional circuitry to help correct for this "series resistance error."

Excised outside-out patch

After obtaining a whole-cell configuration, withdrawal of the pipette pulls a small area of membrane away from the cell. This region of membrane eventually detaches from the cell and seals itself. This membrane patch is oriented with the outer membrane leaflet toward the outside of the pipette. This configuration is used for ligand-gated ion channel studies utilizing rapid switching of the external solution to create an "artificial synapse."

Related Links

See Patch Clamp Technique

 

 

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Critical Care and Pain Medicine
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