Pflügers Arch - Eur J Physiol (2001) 441:425–433 DOI 10.1007/s004240000448
O R I G I N A L A RT I C L E
Xinmin Xie · Timothy J. Dale · Victoria H. John Heather L. Cater · Timothy C. Peakman Jeffrey J. Clare
Electrophysiological and pharmacological properties of the human brain type IIA Na+ channel expressed in a stable mammalian cell line Received: 26 May 2000 / Received after revision: 19 August 2000 / Accepted: 31 August 2000 / Published online: 8 November 2000 © Springer-Verlag 2000
Abstract The human brain voltage-gated Na+ channel type IIA α subunit was cloned and stably expressed in Chinese hamster ovary cells and its biophysical and pharmacological properties were studied using wholecell voltage-clamp. Fast, transient inward currents of up to –8000 pA were elicited by membrane depolarization of the recombinant cells. Channels activated at –50 mV and reached maximal activation at –10 mV to 0 mV. The reversal potential was 62±2 mV which is close to the Na+ equilibrium potential. The half-maximal activation and inactivation voltages were –24±2 mV and –63± 1 mV, respectively. Currents were reversibly blocked by tetrodotoxin with a half-maximal inhibition of 13 nM. The effects of four commonly used anti-convulsant drugs were examined for the first time on the cloned human type IIA channel. Lamotrigine and phenytoin produced concentration- and voltage-dependent inhibition of the type IIA currents, whereas, sodium valproate and gabapentin (up to 1 mM) had no effect. These results indicate that recombinant human type IIA Na+ channels conduct tetrodotoxin-sensitive Na+ currents with similar properties to those observed in recombinant rat brain type IIA and native rat brain Na+ channels. This stable cell line should provide a useful tool for more detailed characterization of therapeutic modulators of human Na+ channels. Keywords Anti-convulsant drugs · CHO cell line · Human type IIA Na+ channel · Patch-clamp recordings · Pharmacological properties T.C. Peakman · J.J. Clare (✉) Molecular Pharmacology Unit, GlaxoWellcome, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts, SG1 2NY, UK e-mail:
[email protected] Tel.: +44-1438-763834, Fax: +44-1438-764488 X. Xie · T.J. Dale · V.H. John · H.L. Cater Neurosciences Unit, GlaxoWellcome, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts, SG1 2NY, UK
Introduction Voltage-gated Na+ channels have a fundamental role in most electrically excitable cells since they conduct the inward currents that occur during the rising phase of action potentials [14]. The main pore-forming component of these channels is the α subunit, and in adult rat brain four different subtypes have been identified, types I, IIA, III and VI [18, 26, 31]. Brain channels also contain three small auxiliary subunits, β1, β2 and β3, which modulate the properties of the channel [16]. Na+ channels are targets for several classes of clinically effective drugs, e.g. anti-convulsants, local anaesthetics, anti-arrhythmics (reviewed by Catterall [4, 5]). Several anti-convulsants that demonstrate clinical efficacy in the treatment of partial and secondary generalized tonic-clonic seizures are suggested to exert their therapeutic effect, at least in part, via modulation of Na+ channels [25, 40]. This includes established drugs such as phenytoin (DPH) and sodium valproate (NaVP), and newer ones such as lamotrigine (LTG) and gabapentin (GBP). Na+ channnel blockers are also being investigated in a number of other therapeutic applications such as analgesia [15, 35] and cerebral ischaemia (reviewed by Taylor and Meldrum [36]). Although the biophysical and pharmacological properties of the rat brain Na+ channels have been well studied, the human brain channels are poorly characterized due to the limited availability of suitable tissue. The development of recombinant systems for the analysis of these channels is therefore an important step in characterizing the activity of established therapeutic agents and understanding their mechanism of action. In the present study, we have developed mammalian cell lines that stably express the human type IIA channel and have characterized its biophysical and pharmacological properties using specific toxins and four currently used anti-convulsant drugs. These results have previously been published in preliminary form [9, 10, 47].
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Materials and methods Cloning and construction of a stable cell line Regions of the human brain types I, IIA and III Na+ channel α subunit cDNAs were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using consensus primers (see [6]) based on the corresponding rat sequences. A mixed probe consisting of these PCR products was then used for hybridization screening of a cDNA library derived from human cerebellum (a gift from Professor I Charles, UCL, London). Two overlapping clones containing the entire coding region of the type IIA subunit were identified. The full-length cDNA was assembled from these two clones and then tailored for expression by replacing the 5′ untranslated region (UTR) with an optimized sequence [19] and removing the 3′ UTR. This construct was then inserted into the expression vector pRDN1 [22], which carries the amplifiable dihydrofolate reductase (DHFR) selection marker, and transfected into the DHFR–ve CHO cell line DUK-B11 [41]. DHFR+ve transfectants were selected in Iscoves MEM medium (Flow-UK) lacking hypoxanthine and thymidine but supplemented with 10% dialysed fetal calf serum and non-essential amino acids (Sigma). To obtain increased expression levels DHFR+ve cell lines were grown in culture medium containing methotrexate (MTX, 0.1 µM) to select for spontaneous amplification of vector copy number [22]. Cells from MTX-resistant cultures were assayed by electrophysiological techniques and the best line was chosen and cloned by two rounds of limiting dilution. Cell culture Clonal cell lines stably expressing the type IIA α subunit were cultured in Iscove’s modified Dulbecco’s medium (Gibco), containing 10% dialysed fetal calf serum (Gibco), non-essential amino acids (Sigma), H-T supplement (hypoxanthine, thymidine; Gibco) and penicillin/streptomycin (Gibco). In addition, the media contained 50 nM MTX (Sigma), to maintain elevated vector copy number, and 200 nM tetrodotoxin (TTX, Sigma), to limit the potential for excessive Na+ influx resulting in cell toxicity. Cells were grown in a 5% CO2 atmosphere with 95% humidity and at 37°C. One to three days prior to electrophysiological recordings the cells were plated on to poly-D-lysine-coated glass coverslips. Whole-cell voltage-clamp recordings Cells grown on glass coverslips were placed into the recording chamber (0.5 ml) and superfused with an extracellular solution at a rate of 2 ml min–1. The extracellular solution contained (mM): NaCl 140, KCl 4.7, MgCl2 1.2, CaCl2 1, glucose 11 and HEPES 5, and the pH was adjusted to 7.4 using NaOH. Patch pipettes were filled with an internal solution consisting of (mM): CsF 120, NaCl 15, Cs-EGTA 10 and HEPES 10, and the pH was adjusted to 7.25 using CsOH. In some experiments, CsF in the internal solution was replaced with CsCl, KCl or K-gluconate and containing 4 mM ATP and 0.5 mM GTP. In all cases the osmolarity of the internal and external solutions ranged 275–290 and 290–310 mosmol/kg, respectively. Patch electrodes were made from borosilicate glass using a Sutter P-97 electrode puller, and when filled with internal solution the patch electrodes were found to have resistances of 2–5 MΩ. Currents were recorded using standard whole-cell voltage-clamp techniques [13], at room temperature (21–23°C) using an Axopatch 200A amplifier and were low-pass Bessel filtered at 5 kHz and digitized at 25–50 kHz. Compensation circuitry was used to minimize 80–85% of the series resistance errors. The cells were maintained at a holding potential (Vh) of –90 mV. Leak currents were subtracted using the P/4 protocol supplied with the pCLAMP6 software, except for the protocols used to demonstrate use dependence in which leak subtraction was switched off. The P/4 leak subtraction protocol did not activate channels since hyperpolarizing pre-pulses were applied that were 1/4 the amplitude
of the test pulse. Membrane potentials were not corrected for junction potentials (
