Voltage-clamp and current-clamp recordings from mammalian DRG neurons

Abstract

We provide here detailed electrophysiological protocols to study voltage-gated sodium channels and to investigate how wild-type and mutant channels influence firing properties of transfected mammalian dorsal root ganglion (DRG) neurons. Whole-cell voltage-clamp recordings permit us to analyze kinetic and voltage-dependence properties of ion channels and to determine the effect and mode of action of pharmaceuticals on specific channel isoforms. They also permit us to analyze the role of individual sodium channels and their mutant derivatives in regulating firing of DRG neurons. Five to ten cells can be recorded daily, depending on the extent of analysis that is required. Because of different internal solutions that are used in voltage-clamp and current-clamp recordings, only limited information can be obtained from recording the same neuron in both modes. These electrophysiological studies help to elucidate the role of specific channels in setting threshold and suprathreshold responses of neurons, under normal and pathological conditions.

Inherited Pain

“…As described previously (19), ganglia were treated with 1 mg/ml collagenase and 0.1 mg/ml protease for 30 min (both from Sigma Aldrich, Germany) and subsequently dissociated using a fire-polished, silicone-coated Pasteur pipette. Neurons were immediately transfected with the hNav1.7 WT or A1632T mutation and EGFP by electroporation with a Nucleofector II (Lonza, Cologne, Germany), plated on poly-D-lysine-coated (200 g/ml, Sigma Aldrich) coverslips and cultured in TNB100 cell culture medium supplemented with TNB100 lipid-protein complex and 100 g/ml streptomycin, penicillin (all from Biochrom, Berlin, Germany).…”

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

“…The external and internal solutions used were as previously described (ref. 49 and see also Supplemental Methods). Pipettes had a resistance of less than 3 MΩ, capacity transients were cancelled, and series resistance was compensated by approximately 90%.…”

Gain-of-function mutation of a voltage-gated sodium channel NaV1.7 associated with peripheral pain and impaired limb development

“…The extracellular solution contained 140 mM NaCl, 3 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM HEPES, and 10 mM dextrose, pH 7.3, adjusted with NaOH (319 mOsm). tetrodotoxin (500 nM) was included in the bath to block endogenous sodium currents in HEK293 cells (27,50). The pipette solution contained the following 140 mM CsF, 1 mM EGTA, 10 mM NaCl, 10 mM HEPES, and 10 mM dextrose, pH 7.3, adjusted with CsOH (adjusted to 311 mOsm with sucrose).…”