2.2 | Injection and voltage-clamp recordings inXenopus oocytes
This study has been conducted conforming the guide for the Care and Use of laboratory Animals (NIH Publication 85-23) and the local ethics commission of the “Regierungspräsidium Giessen” approved the experiments using Xenopus frogs (Nasco, Wisconsin, USA) (MR 20/28 Nr. A 23/2017). cRNA for wild-type Kv1.5 or mutant Kv1.5 constructs were injected into isolated stage IV and V Xenopus laevis oocytes as described previously (Streit, Netter et al. , 2011). The oocytes were cultured in bath solution including (in mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES; pH 7.5 supplemented with 50 mg/L gentamycine, 274 mg/L sodium pyruvate and 88 mg/L theophylline at 18°C for 1-13 days before the experiments. All measurements were performed using standard two microelectrode voltage clamp techniques (Stühmer, 1992). Measurements were recorded at room temperature (21–23°C) with a TurboTEC 10 CD amplifier (npi electronic, Tamm, Germany) and a Digidata 1200 Series A/D converter (Molecular Devices, California, USA). Micropipettes with a resistance of 0.5–1.2 MΩ when filled with 3 mM KCl were made from borosilicate glass capillaries GB 150TF-8P (Science Products, Hofheim, Germany) and pulled with a DMZ-Universal Puller (Zeitz, Martinsried, Germany). R/S-bupivacaine and ropivacaine were prepared as a 125 mM stock solution in DMSO, stored in a lightproof manner and added to the bath solution just before the recordings. The holding potential was -80 mV. To guarantee a fully regeneration from inactivation a minimum interpulse interval of 10 s was chosen. The inhibition was calculated at end of a voltage step to +40 mV for 2 s. Endogenous current component of the oocytes in amount of 150 nA has been subtracted in the analysis to increase the sensitivity of scan. The ratioI drug/I ctrl was also determined using this protocol. The current corresponding to the first pulse after a 12 min pulse free period in the presence of drug was divided by the last trace in absence of drug before the pulse free period. The IV -protocol which was used to obtain the current-voltage relationship (IV ) comprises 10 mV steps in voltage, ranging from -70 mV to +70 mV for 200 ms, and a final step to -40 mV for 300 ms. A Hill plot was used to calculate the half maximal inhibitory concentration (IC50). Boltzmann plot was used to calculate the voltage at half maximal activation (V1/2).