Xenopus Oocyte Electrophysiology
Eggs (oocytes) from female African clawed frogs (Xenopus laevis) have been used for several decades as an expression system for a variety of ion channels, transporters, and receptors. The advantages of this system are the large size of the cells, which makes them easy to work with and also enables them to produce large numbers of ion channels, which can then produce large currents for measurement.
Xenopus oocytes are up to 1 mm in diameter and are characterized by a pigmented animal pole and a pale vegetal pole. The animal pole contains the nucleus and cytoplasmic machinery for protein production, while the vegetal pole is primarily a source of energy during early embryogenesis. Ooctyes are harvested from anesthetized females via a mini-laparotomy and treated with collagenase enzymes to digest connective tissues that keep the oocytes bound together in ovarian lobes.
For heterologous expression of proteins, xenopus oocytes can be micro-injected with either cDNA or mRNA encoding protein or subunits. cDNA injection is into the nucleus, while mRNA can be injected into the cytoplasm. In general, expression of these exogenous genes occurs in 24-72 hours.
Figure 3: Injection of mRNA into Xenopus oocytes
Two micro-electrode electrophysiology (Figure 4) is used to measure currents from Xenopus oocytes, which have magnitudes ranging from 20 nA up to 20 ľA. The electrodes used to impale the ooctye are actually glass capillary pipettes pulled to a fine tip and filled with 3 M KCl. These are mounted over silver wires that have been treated to produce a coating of AgCl. Thus, chloride ions are the major conductive element within the electrodes. Two electrodes are used in order to minimize the series resistance correction that would be produced when passing large currents through a single high resistance electrode. One electrode is used to monitor the voltage (V) inside the oocyte relative to a ground electrode in the surrounding bath solution. In voltage-clamp mode, this voltage is compared to a set ?clamp? value and current (I) is applied via a second electrode in order to minimize the difference between these values. The voltage-clamp electrometer circuitry measures the current flowing through this second electrode and produces a voltage signal that is proportional to the current.
When studying ligand-gated ion channels, oocytes are useful for qualitative or comparative measurements. However, solution exchange, which is required to activate these channels, is difficult to achieve in less than several hundred milliseconds. As a result, many ligand-gated ion channels may desensitize before solution exchange is complete in these experiments. This creates inaccuracies in the relative magnitudes of currents under different experimental conditions and makes oocytes inappropriate for measuring the rates of agonist-induced state transitions. In some cases, where desensitization is relatively slow, Xenopus oocytes may provide accurate data that is appropriate for quantitative analysis and pseudo-equilibrium modeling.
Figure 4: Two electrode electrophysiology
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