| Assay Method Information | |
| | patch clamp assay |
| Description: | Inhibition of the T-type voltage gated calcium channel (Cav3.1) was evaluated using a HEK-293 natClytin/TASK1+Cav3.1 cell line. Currents were recorded using the SyncroPatch 384PE automated, patch clamp system. Pulse generation and data collection were performed with PatchController384 V1.3.0 and DataController384 V1.2.1 (Nanion Technologies). Off-line analysis was performed using Excel and Graphpad Prism (V 8.4.2) with complete data files uploaded to Dotmatics. The access resistance and apparent membrane capacitance were estimated using built-in protocols. Current was recorded in whole cell configuration from a population of cells. The cells were lifted, triturated, and resuspended at 800,000 cells/ml. The cells were allowed to recover in the cell hotel prior to experimentation. Currents were recorded at room temperature. The external solution contained the following (in mM): NaCl 80, NMDG 60, KCl 4, MgCl2 1, CaCl2 6, glucose 5 and HEPES 10 (pH=7.4, Osmolarity ˜300 mOsm). The extracellular solution was used as the wash, reference and compound delivery solution. The internal solution contained the following (in mM): CsF 110, CsCl 10, NaCl 10, EGTA 10, HEPES 10 (pH=7.2, Osmolarity ˜295 mOsm). The compound plate was created at 2× concentrated in the extracellular solution. The compound was diluted to 1:2 when added to the recording well. The amount of DMSO in the extracellular solution was held constant at the level used for the highest tested concentration. For the voltage clamp experiments on Cav3.1, data were sampled at 10 KHz. After establishment of the seal and the passage in the whole cell configuration, the cells were held at −120 mV. Cav3.1 current was evoked using a 100 ms step to −20 mV (to measure resting state block), followed by a 1600 ms step to −65 mV and a second 100 ms step to −20 mV (to measure voltage dependent block). The voltage protocol was applied every 15 seconds in the absence and in the presence of the compounds under investigation. 2.5 mM Nickel was used to completely inhibit Cav3.1 current to allow for offline subtraction of non-Cav3.1 current. Current amplitude (pA) was measured in the peak 1 and 2. The average of last 3 sweeps of each liquid period (vehicle, compound under investigation, full block) was calculated. Nickel-sensitive current was used to calculate the % of inhibition in the presence of the compound under investigation. |
| Affinity data for this assay | |
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