Two-Electrode Voltage Clamp of Xenopus Laevis Oocytes Workshop

Basics of voltage clamp
The voltage clamp technique is used to measure ionic currents in response to precisely controlled changes in the transmembrane potential of an isolated cell. Large cells (e.g., frog oocytes) can be studied using two-electrode voltage clamp (Figure 1). The oocyte is impaled by two glass microelectrodes, one for voltage sensing and one for current injection. The transmembrane potential is measured by the voltage-sensing electrode (V1) connected to a high input impedance amplifier (Amp 1). This signal is compared to a command voltage generated by a computer at the input of Amp 2. The output of the high gain feedback Amp 2 is a current delivered to the cell interior by the second micropipette. This current is sufficient to force the transmembrane potential to equal the command voltage. The current delivered by micropipette 2 is monitored as “I2” via a current-to-voltage converter. In this lab exercise you will observe recordings of whole cell ionic currents conducted by an “unknown” ion channel heterologously expressed in Xenopus oocytes using the two-electrode voltage clamp technique. A Warner 0C-725C amplifier (Figure 2) will be used to record currents in response to changes in membrane voltage in oocytes that were injected 24 hours previously with cRNA encoding the “unknown” ion channel. Data acquisition will be performed using a personal computer and an Axon low-noise 1440A analog-to-digital (A/D) interface.
2980690374650290830025400290830025400306070063500Fig. 2: Warner OC-725C oocyte clamp amplifier306070063500
Microelectrodes
Microelectrodes are pulled from 1.0 mm (external diameter) borosilicate glass tubing using a Narishighe PC-10 vertical micropipette puller. The method used by this puller is to stretch the glass capillary vertically using the gravitational force of its own and added weights, thereby thinning the glass tubing until it breaks resulting in two glass microelectrodes with a tip diameter of ~1 µm and a tip resistance of 0.2 - 1 MΩ. Microelectrodes are backfilled with a 3M KCl solution using a special syringe needle and one electrode secured into each of the two electrode holders, making sure that Ag/AgCl wire makes contact and therefore electrical connection with KCl solution in the pipette.
Preparation of oocytes
Ovaries are obtained from Xenopus laevis (Figure 3) killed humanely in accordance with regulations issued by the Home Office of the United Kingdom under the Animals (Scientific Procedures) Act, 1986. Oocytes are manually dissected into clumps of ~10 oocytes with forceps and washed in sterile, calcium-free, OR-2 solution containing (in mM) 82.5 NaCl, 2.5 KCl, 1 MgCl2, 10 HEPES (pH 7.4 with NaOH). Oocytes are then rotated in OR-2 solution containing 2 mg/ml collagenase type II at room temperature for ~60-90 minutes to dissociate oocytes and remove the follicular layer (Figure 4). Oocytes are then washed with calcium containing ND96 storage solution containing (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, 1% penicillin/streptomycin solution, 0.1% gentamycin (pH 7.6 with NaOH) and stored at 16 °C in a refrigerated incubator.
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