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| | Voltage-Clamp Assay |
| Description: | All experiments were conducted at room temperature (22-24° C.) with an EPC-10 amplifier and Pulse software (HEKA, Lambrecht/Pfalz, Germany) in the whole-cell mode of the patch-clamp technique. Human embryonic kidney (HEK)-293 cell lines stably expressing hNav1.1, hNav1.5, hNav1.7 channels (generously provided by Dr. Christopher Lossin, University of California Davis), hNav1.4 (Frank Lehmann-Horn, University of Ulm), or hKv2.1 channels (James Trimmer, University of California Davis) were bathed in extracellular solution containing (in mM): 160 NaCl; 4.5 KCl; 1 MgCl2; 2 CaCl2; 10 HEPES [pH was adjusted to 7.4 using NaOH (310 mOsm)]. Pipettes were filled with intracellular solution containing (in mM): 145 KF; 2 MgCl2; 10 ethylene glycol tetraacetic acid; 10 HEPES (pH adjusted to 7.2 with KOH; 300 mOsm). Neuroblastoma N1E-115 cells (ATCC, Manassa, Va., USA) expressing Nav1.2 were patched with a CsF internal solution consisting of (in mM): 10 NaF; 110 CsF; 20 CsCl; 2 ethylene glycol tetraacetic acid; 10 HEPES (CsOH to pH 7.35; 300 mOsm). All pipette tip resistances were 2-4 MΩ. Series resistances of 3-10 MΩ were compensated 40-80%. All cells were voltage-clamped to a holding potential of −90 mV unless otherwise specified. The sampling frequency was 5 kHz. Na+ currents were elicited by 30-ms pulse to 0 mV from −90 mV applied every 10 s. Kv2.1 currents were elicited by 200-ms voltage steps from −90 to 40 mV applied every 10 s. HEK-293 or COS-7 cells stably expressing hKCa2.1, rKCa2.2, and hKCa2.3 have been described previously (Sankaranarayanan et al., 2009). Cells were held at −80 mV and KCa currents elicited by dialysis with a K+ aspartate based internal containing 250 nM free Ca2+ (pH 7.2, 290 mOsm, pipette resistance 1.5 MΩ). To reduce currents from native chloride channels, Na+ aspartate Ringer was used as an external solution. KCa2 currents were recorded with 200-ms voltage ramps from −120 to +40 mV applied every 10 s, and the fold increase of slope conductance at −80 mV by drug was taken as a measure of channel activation. Data analysis, fitting, and plotting were performed with IGOR-Pro (Wavemetrics, Lake Oswego, Oreg., USA) and Origin 9.0 (OriginLab, Northampton, Mass., USA). |
| Affinity data for this assay | |
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