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

Department of Pharmacology and Toxicology, Stark Neurosciences Institute, Indiana University School of Medicine, Indianapolis, IN, USA.
Nature Protocol (Impact Factor: 9.67). 02/2009; 4(8):1103-12. DOI: 10.1038/nprot.2009.91
Source: PubMed


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.

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    • "However, a time window of 10 s is considered to be relevant for inducing slow inactivation (Estacion et al. 2010), and therefore, differences seen in mutated Nav1.7 channels compared to WT are representative of enhanced slow inactivation. To investigate the time to recovery from fast inactivation, first a 20-ms pulse to -10 mV was applied followed by a recovery phase between 5 and 100 ms and a second pulse to -10 mV (Cummins et al. 2009). Recorded peak currents were then normalized and fitted using a single exponential equation (one-phase association). "
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    ABSTRACT: We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 (DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype–phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM. Electronic supplementary material The online version of this article (doi:10.1007/s12017-012-8216-8) contains supplementary material, which is available to authorized users.
    Neuromolecular medicine 01/2013; 15(2). DOI:10.1007/s12017-012-8216-8 · 3.68 Impact Factor
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    • "1.5–3 M) was filled with a pipette solution composed of KCl (140 mM), CaCl 2 (1 mM), MgCl 2 (2 mM), EGTA (11 mM), Hepes (5 mM) and dextrose (10 mM) titrated to pH 7.3 with KOH (304 mosmol l −1 ). The cells were continuously superfused with solution; composed of (mM): 140 NaCl, 3.0 KCl, 2.0 MgCl 2 , 2.0 CaCl 2 , 10.0 Hepes and 10 dextrose, titrated to pH 7.3 with NaOH (Cummins et al. 2009 "
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    The Journal of Physiology 11/2010; 588(Pt 21):4303-15. DOI:10.1113/jphysiol.2010.192971 · 5.04 Impact Factor
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    ABSTRACT: Blockade of the human ether-a-go-go-related gene (hERG) potassium channel, with a consequent possibility of QT prolongation and increased susceptibility to a characteristic polymorphic ventricular arrhythmia, torsade de pointes, is an important cause of withdrawal of drugs from the market. In the aftermath of recent drug withdrawals, regulatory agencies now require in vitro hERG screening of all pharmaceutical compounds that are targeted for human use. To minimize the potential for failure in later-stage drug development, many pharmaceutical and biotechnology companies have begun to use automated patch clamp systems with higher throughput than conventional manual patch-clamp techniques to conduct routine functional hERG screening during drug discovery and early development. We have optimized an automated patch-clamp hERG screening method for the PatchXpress 7000A system (Molecular Devices, Sunnyvale, CA) using potassium fluoride (KF) in the internal recording solution. In this study we show that (1) the biophysical and pharmacological properties of hERG current recorded with KF are similar to those with standard potassium chloride solutions, (2) use of KF significantly improves the success rate of hERG screening using PatchXpress without compromising data quality, and (3) utilization of KF can significantly increase the throughput of hERG screening with PatchXpress.
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