Whole-cell patch-clamp recordings of Ca2+ and Na+ currents in acutely dissociated DRG neurons
Recordings were obtained from acutely dissociated DRG neurons as described earlier (Bellampalli et al., 2019). Patch-clamp recordings were performed at room temperature (22–24°C). Currents were recorded using an EPC 10 Amplifier-HEKA (HEKA Elektronik, Ludwigshafen, Germany) linked to a computer with Patchmaster software. DRG neurons were incubated overnight (~16-24 h) with 20 µM of CBD3063.
For total calcium current (ICa2+) recordings, the external solution consisted of the following (in mM): 110 N-methyl-D-glucamine, 10 BaCl2, 30 TEA-Cl, 10 HEPES, 10 glucose, 0.001 TTX (pH 7.29 adjusted with TEA-OH, and mOsm/L= 310). Patch pipettes were filled with an internal solution containing (in mM): 150 CsCl2, 10 HEPES, 5 Mg-ATP, and 5 BAPTA, (pH 7.2 adjusted with CsOH, and mOsm/L= 305). Peak Ca2+2+channels, DRGs were treated with a Cav inhibitor cocktail omitting the inhibitor specific to the subtype being tested (e.g., to measure Cav2.2 currents, ω-conotoxin GVIA is omitted): Nifedipine (10 µM, L-type), ω-Conotoxin-GVIA (500 nM, P/Q-type) (Feng, Hamid, Doering, Bosey, Snutch & Zamponi, 2001), SNX482 (200 nM, R-type) (Newcomb et al., 1998), ω-agatoxin (200 nM, P/Q-type) (Mintz, Venema, Swiderek, Lee, Bean & Adams, 1992), TTA-P2 (1 µM, T-type) (Choe et al., 2011).
For Na+ current (INa+) recordings, the external solution contained (in mM): 130 NaCl, 3 KCl, 30 tetraethylammonium chloride, 1 CaCl2, 0.5 CdCl2, 1 MgCl2, 10 D-glucose and 10 HEPES (pH 7.3 adjusted with NaOH, and mOsm/L= 324). Patch pipettes were filled with an internal solution containing (in mM): 140 CsF, 1.1Cs-EGTA, 10 NaCl, and 15 HEPES (pH 7.3 adjusted with CsOH, and mOsm/L= 311). Peak Na+
To isolate potassium currents (IK+), DRG neurons were bathed in external solution composed of (in millimolar): 140 N-methyl-glucamine chloride, 5 KCl, 1 MgCl2, 2 CaCl2, 10 D-glucose and 10 HEPES (pH adjusted to 7.3 with KOH and mOsm/L= 313). Recording pipettes were filled with internal solution containing (in mM): 140 KCl, 2.5 MgCl2
Normalization of currents to each cell’s capacitance (pF) was performed to allow for collection of current density data. For I-V curves, functions were fitted to data using a non-linear least squares analysis. I-V curves were fitted using double Boltzmann functions: