HEK293 cells (Robert S Kass laboratory, Columbia University, New York) were transfected by Lipofectamine LTX (Invitrogen) according to the manufacturer‘s instructions. The plasmids used were the 2019 amino acid isoform of the mouse voltage-gated sodium channel (pcDNA3-mNa _v1.5; a gift from Thomas Zimmer, University of Jena, Germany ⁶ ), human Na _v1.5 (pcDNA3.1-hNa _v1.5), and three differently tagged hNa _v1.5 (pcDNA3.1-hNa _v1.5-GFP-N-terminal, pEYFP-hNa _v1.5, and pcDNA3.1-FLAG(299/300)-hNa _v1.5). The FLAG-tag is an eight amino acid-long epitope (DYKDDDDK) that was inserted into the extracellular loop linking the transmembrane segments S5 to S6 of domain I, between the residues Leu-299 and Val-300; GFP and YFP were previously added by T. Zimmer to the N-terminal ⁷ . For wild-type and tagged hNa _v1.5, 1 µg of one of the listed plasmids, 1 µg of empty pcDNA3.1 (Invitrogen), and 0.4 µg of DNA coding for CD8 (Robert S Kass laboratory) was used for transfection. In order to measure currents of comparable size, 0.01–1 µg of mNa _v1.5 was co-transfected with 1 µg of empty pcDNA3.1, and 0.4 µg of DNA coding for CD8. Transfected HEK293 cells were then grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% calf serum (Gibco), 0.2% glutamine (Sigma), and 20 mg/ml gentamycin (Gibco), and incubated at 37°C with 95%O ₂/5%CO ₂.

All experiments were performed in the whole-cell voltage-clamp mode. The extracellular solution contained (in mM): 50 NaCl, 80 NMDG-Cl, 5 CsCl, 2 CaCl ₂, 1.2 MgCl ₂, 10 HEPES, 5 Glucose, adjusted to pH 7.4 with CsOH, and with an osmolality of 280–290 mOsm. The internal solution consisted of (in mM): 70 CsAsp, 60 CsCl, 1 CaCl ₂, 1 MgCl ₂, 10 HEPES; 11 Cs ₂EGTA, 5 Na ₂ATP, adjusted to pH 7.2 with CsOH, and with an osmolality of 297 mOsm. Recordings were performed at room temperature (20–22°C) using a VE-2 amplifier (Alembic Instruments, Montreal, Canada). Data was acquired by Clampex 10.2 (Axon Instruments, Union City, Canada). Membrane resistance was ≥ 1 GΩ and access resistance ≤ 6.1 MΩ. Transfected cells were recognized by the addition of 1 µl/ml Dynabeads CD8 (Invitrogen) into the extracellular solution. Current-voltage (I/V) curves` were assessed by depolarising cells from a holding potential of -100 mV to voltages of between -80 and 40 mV during 20 ms. Steady-state inactivation properties were measured by the following protocol: the cells were kept at a holding potential of -100 mV and then hyper- and depolarised during 500 ms to voltages of between -120 and 0 mV in steps of 5 mV, followed by 20 ms at the voltage that elicited the maximal response during the I/V-protocol. Voltage-dependent activation was read either from the I/V- or the steady-state inactivation-protocol. To characterise the recovery from inactivation, the cells were depolarised from a holding potential of -100 mV for 100 ms, repolarised to -100 mV at a recovery time of 0.25–3000 ms, and depolarised again for 25 ms. By varying the time of the first depolarisation step from 3 to 3000 ms followed by 25 ms of repolarisation, the onset of slow inactivation was determined (see insets of Figure 2 and Figure 4).

Peak values for all protocols were detected and measured by Clampfit 10.2 and I/V-relationships were fitted using KaleidaGraph 3.5 (Synergy Software, Reading, USA). Values were normalised to membrane capacitance. The following formula was used to fit I/V-curves and to calculate reversal potentials: I _Na = (G _max(V-V _rev,Na))/(1+e ^V-V0.5/K) with I _Na = sodium current in pA, G _max = max. conductance = 60 Ω ^-1, V _rev,Na = reversal potential = 40 mV, K = (-zδF)/FR = equilibrium constant = -5, V _0.5 = voltage for 50% of maximum current = -20 mV. Activation and inactivation curves were fitted with the Boltzmann equation f ₀ = 1/(1+ e ^V-V0.5/K) with f ₀ = fraction of open channels/total available channels. Statistical analyses were performed using two-tailed Student's t-tests. A p value <0.05 was considered statistically significant.

To compare the biophysical properties of the cardiac sodium channel Na _v1.5 from the human (hNa _v1.5) or the mouse sequence (mNa _v1.5), we measured the electrophysiological properties of hNa _v1.5 and mNa _v1.5, transiently expressed in HEK293 cells. Representative I _Na recordings are shown in Figure 1. The responses to all applied protocols revealed similar characteristics for both channels, except for the reversal potential and the slope of steady-state inactivation ( Figure 2 and Table 1). The peak currents from the I/V-protocol were at -15 mV for both channels ( Figure 2A). Furthermore, activation and inactivation of 50% of the channels occurred for both channels at ~-28 mV and ~-71 mV, respectively. In addition, the slopes of the activation curve were comparable for both channels (6.00 mV/e-fold in human and 6.24 mV/e-fold in mouse). Significant differences could be detected in the reversal potential V _rev (51.0 mV and 56.6 mV, P<0.01) and in the slope of the inactivation curve (5.95 mV/e-fold and 6.67 mV/e-fold, P<0.01) ( Figure 2B). In addition, mNa _v1.5 had a tendency to recover faster from inactivation ( Figure 2C). The fraction of channels entering into a slow inactivation state was similar for both channel types ( Figure 2D).

The second set of experiments addressed the effects of adding epitopes to Na _v1.5 on its biophysical properties. To do this, we assessed the influence of these epitopes on I _Na by expressing differently tagged hNa _v1.5 in HEK293 cells and performing whole-cell voltage-clamp experiments similar to those described above. YFP- and GFP-tags were added to the N-terminus; the FLAG-tag was inserted into the extracellular loop linking S5 to S6 of domain I, between residues Leu-299 and Val-300. Representative I _Na recordings for all transfected constructs are shown in Figure 3 and the data is summarised in Table 2. With the exception of the GFP-tagged construct, tagging of hNa _v1.5 led to a significant decrease in peak current I _max ( Figure 4A) compared to the control WT hNa _v1.5 (FLAG: 57 pA/pF with P<0.01, YFP: 120 pA/pF with P<0.05, WT hNa _v1.5: 240 pA/pF). Adding GFP did not affect any of the biophysical properties of the human sodium channel, while a shallower activation slope (6.87 vs. 5.91 mV/e-fold, P<0.05, Figure 4B and Table 2) was observed for the YFP-tagged channel. The most pronounced effects were observed for the FLAG-tagged hNa _v1.5. The activation slope was significantly shallower (6.96 vs. 5.91 mV/e-fold, Figure 4B and Table 2), indicating that the activation of this channel is less sensitive to voltage changes. In addition, the V _1/2 of activation was shifted towards more positive voltages by about 5 mV, with -23.9 mV in FLAG-hNa _v1.5, compared to -28.9 mV in untagged hNa _v1.5. Finally, the reversal potential was decreased in the FLAG-hNa _v1.5 (FLAG 39.3 mV and untagged 51.8 mV, Figure 4B). Recovery from inactivation ( Figure 4C) and onset of slow inactivation ( Figure 4D) were comparable for all channels.