| Assay Method Information | |
| | Human Nav1.7 Inhibitory Activity Evaluation |
| Description: | The amount of current through human Nav1.7 was recorded using a fully automatic patch clamp device QPatch16X (Sophion Biosciences) at room temperature. As the extracellular solution, the solution of 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) at pH 7.4 was used. As the intracellular solution, the solution of 135 mM CsF, ⅕ mM glycol ether diaminetetraacetic acid (EGTA)/CsOH, 10 mM HEPES, 10 mM NaCl at pH 7.3 was used. The test compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted with the extracellular solution to attain the DMSO concentration of 0.1% at the time of assay. The current response was obtained at a sampling frequency of 25 kHz, and noise was removed with a 3 kHz low-pass filter. The holding potential was −100 mV. Correction of the leakage current was carried out by applying a step pulse of −120 mV before the test pulse. In order to investigate inhibitory action of the test compound, the following test pulse was given: Namely, after depolarizing pulse of −10 mV was applied for 50 milliseconds, it was fixed at −120 mV for 500 milliseconds; a potential at which about 30-40% of the channel was inactivated was maintained for 15 seconds, then fixed again at −120 mV for 100 milliseconds, and finally a −10 mV depolarization pulse was given for 50 milliseconds. Test pulses were given before and after the test compound addition. |
| Affinity data for this assay | |
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