Materials and methods
Molecular biology. Template cDNAs encoding hKir2.x, hERG, hKv1.5, hKv4.3, hKCNQ1, hKCNE1 and hGrb were linearized with an appropriate restriction enzyme (New England Biolabs, MA, USA) and cRNA was synthesized from 1 μg of linearized DNA by in vitro Staurosporine (mMessage mMachine T7 kit or mMessage mMachine SP6 kit, Ambion, Applied Biosystems, Germany). cRNA concentrations were evaluated by photospectrometry and transcript quality was verified by agarose gel electrophoresis.
Oocyte isolation. Ovarian lobes were harvested from Xenopus laevis frogs anesthetized with a 0.17% tricaine solution. Oocytes were treated for 120 min with collagenase (1 mg/ml, Worthington, type II) in ND96-Ca2+-free solution containing (in mM): 96 NaCl, 2 KCl, 2 MgCl2, 5 HEPES (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid) (pH 7.6) to remove follicle cells, and then stored at 17 °C in ND96 containing gentamycin (50 mg/L), theophylline (0.5 mM) and Na-pyruvate (2.5 mM). For characterization of cardiac K+ channels, each oocyte was injected with 1 ng Kir2.x cRNA or 4 ng cRNA encoding the other channels followed by coinjections with 3 ng cRNA encoding Grb7 or 10.
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