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: