Expression of Human Cardiac Sodium Channel a and B Subunits

Supplement

Cell culture. Human kidney epithelial cells tsA201 were cultured in DMEM medium supplemented with streptomycin (100μg/ml), penicillin (100 units/ml), and 10% bovine serum in a 5% C02 /room air humidified atmosphere at 370C.

Sodium channel α and β subunit clones DNA constructs. The method of preparation of cDNA in our laboratory is standard and has been previously described [1]. The human Nav1.5 (former hH1a) was kindly provided by Drs. H. Hartmann and A.M. Brown (Baylor University, Houston, TX). The human cardiac β1 and β2 subunits were cloned in pcDNA3.1 vector from a human heart poly (A)+ RNA using RT-PCR. Also β-GFP construct was prepared for the fluorescence control of expression. In this case the cDNA sequences encoding of the β1 and β2 subunits were subcloned into the mammalian expression vector pcDNA3.1/CT-GFP (Invitrogen) and sequenced.

Transient expression. Cells (tsA201) were transiently transfected by Nav1.5 alone and/or simultaneously with human cardiac β1 or β2 subunits using a calcium-phosphate procedure by Lipofectin\Lipofectamine (Gibco BRL). The expression has been done in two ways: 1) the cDNA ratio for the α and β-GFP was 1:1, and 2) 1:5 (as previously suggested [2]) when cDNA encoding β only (without GFP, ) The transfection and cell culturing procedure was similar to that recently described [3]. The transfected cells were cultured for ~24h prior to patch-clamp studies.

Patch-Clamp . Whole-cell recordings were performed using pClamp 8.0 (Axon Instruments Inc., Foster City, CA) software and an Axon instruments Axopatch 200 patch clamp at room temperature (22-24 oC), low pass filtered (-3dB, 5kHz), and sampled at 20kHz. Pipettes were pulled from borosilicate glass capillaries (K150F, WPI Inc., Sarasota, Fl) and tips were fire polished. The whole-cell transient (INaT) and late Na+ current, INaL, was measured as previously described [4]. The resistance of patch pipettes was ~1.2 MΩ, and total pipette-cell resistance was >5GΩ. The leak current was obtained after TTX (25 μM) application and was subtracted from the current traces. Adjustment of series resistance compensation was made just before recording to provide optimum voltage control [5]. INaL was assessed by 2 s membrane depolarizations from a holding potential (Vh) of -140 mV to applied with a stimulation frequency of 0.1 Hz. To improve the resolution, 20-50 traces were averaged. Extracellular solution contained (here and below in mM): 140 NaCl, 5 CsCl, 1.8 CaCl2, 2 MgCl2, 5 glucose, 0.002 nifedipine, 5 HEPES-CsOH buffer (pH 7.3). Pipette solution contained: 5 NaCl, 133 CsCl, 2 MgATP, 20 tetraethylammonium chloride, 10 EGTA, 5 HEPES-CsOH buffer (pH 7.3).

REFERENCES

1.MakielskiJC, LimberisJT, ChangSY, FanZ and KyleJW. Coexpression of beta 1 with cardiac sodium channel alpha subunits in oocytes decreases lidocaine block.Molecular Pharmacology 1996; 49:30-9 Medline.

2.QuY, RogersJC, ChenSF, McCormickKA, ScheuerT and CatterallWA. Functional roles of the extracellular segments of the sodium channel alpha subunit in voltage-dependent gating and modulation by beta1 subunits.J Biol Chem. 1999; 274:32647-54 Medline. doi:10.1074/jbc.274.46.32647

3.MaltsevVA, KyleJW, MishraS and UndrovinasA. Molecular identity of the late sodium current in adult dog cardiomyocytes identified by Nav1.5-antisense inhibition.Am J Physiol Heart Circ Physiol 2008; 295:H667-H676 Medline. doi:10.1152/ajpheart.00111.2008

4.MaltsevVA, SabbahHN, HigginsRSD, SilvermanN, LeschM and UndrovinasAI. Novel, ultraslow inactivating sodium current in human ventricular cardiomyocytes.Circulation 1998; 98:2545-2552 Medline.

5.MaltsevVA and UndrovinasAI. Cytoskeleton modulates coupling between availability and activation of cardiac sodium channel. Am J Physiol 1997; 273 (Heart Circ. Physiol. 42): H1832-H1840.