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Introduction The giant patch or “macropatch” technique was originally developed to measure currents of electrogenic transporters or pumps, which are far smaller than those carried by ion channels (1, 2). Large excised patches are also very useful for the study of ion channels. Generally this technique gives similar information to that obtained by using single-channel measurements in excised patches. It does not give the detailed information that single-channel measurements give on gating kinetics, conductance, etc., but it is a lot more robust, and simpler to analyze, as it gives composite current of hundreds or even thousands of channels. The excised inside-out configuration allows the perfusion of various compounds to the intracellular surface of the patch membrane, thus the study of the effects of intracellular regulators in isolation from the cellular environment. This chapter is based on our experience with this technique with inwardly rectifying K+ (Kir) channels and various transient receptor potential (TRP) channels (3–8).

Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_9, © Springer Science+Business Media, LLC 2013

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Tibor Rohacs

Xenopus laevis oocytes have been used for quite a long time as an expression system to study recombinant ion channels (9). There are many articles that provide excellent description of oocyte preparation and RNA injection (10); we will provide a brief description here based on our experience.

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Materials

2.1 Oocyte Preparation and Injection

1. OR2 solution for oocyte preparation and storage. The composition of OR2 is 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH = 7.4. We designate this Ca2+-free solution as OR2(−). Since large volumes are used because of the repetitive wash steps during oocyte isolation, it is practical to prepare a 20× concentrate, and dilute several liters to working concentration to keep in a large bottle. After dilution the pH needs to be adjusted, and the solution can be kept at 4°C for several weeks; it is not necessary to sterile filter the OR2(−) solution. After digestion, the eggs are kept in OR2 solution with an additional 1.8 mM Ca2+ and penicillin-streptomycin (designated as OR2(+) solution); we recommend that this solution is sterile filtered. 2. Collagenase solution. We use 0.1–0.2 mg/mL of type IA collagenase from Sigma. We dissolve it freshly in OR2(−) just before starting the digestion process (see Note 1). 3. MS222 solution. 0.25% Ethyl 3-aminobenzoate methanesulfonate (tricaine, MS222) dissolved in H2O, pH adjusted to ~7.4. We prepare 1 L, which can be reused several times, when kept in a cold room. 4. Scissors, forceps. 5. Sutures (Chromic gut). 6. Oocyte incubator (18°C) preferably with an electrical outlet inside to power the rotating platform. 7. Dissecting microscope and light source. 8. Injector (Drummond Nanoject). 9. Nuclease-free water and mMessage Machine kit (Ambion).

2.2 Patch Clamp Measurements

We recommend that all solutions a