Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Na_v1.5 expressed in HEK293 cells

Introduction

The voltage-gated cardiac sodium channel Na_v1.5 is responsible for the initial phase of the cardiac action potential and plays a central role in cardiac impulse propagation¹. Its role in human disorders has been underlined by the findings of several hundred mutations in its gene, SCN5A, that are linked to inherited cardiac electrical disorders such as congenital long QT syndrome and Brugada syndrome². In recent years, it has been demonstrated that Na_v1.5 interacts with and is regulated by different proteins (recently reviewed by Shy et al.³). Many of these interacting proteins were also found to be mutated in patients with genetically-determined cardiac arrhythmias⁴. The generation of genetically-modified mouse models, harbouring mutations in the Scn5a gene, has proven to be a very informative approach to investigate the various human phenotypes that are linked to the genetic variants of this gene⁵. Since Na_v1.5 interacts with many proteins during its life cycle in cardiac cells, it would be of great interest to generate a mouse model that permits the biochemical purification of Na_v1.5 with high efficiency, hence allowing the co-purification of interacting proteins. The identity of these co-purified proteins may then be determined by using mass spectrometry-based technologies. In order to do this, one needs to first generate a knock-in mouse model, where a high-affinity epitope would be added to the mouse Scn5a gene that codes for Na_v1.5.

The goals of this short study were (1) to compare the biophysical properties of the sodium current (I_Na) generated by mouse Na_v1.5 and human Na_v1.5 constructs expressed in HEK293 cells, and (2) to investigate the possible alterations of the biophysical properties of human Na_v1.5 constructs that were modified with specific epitopes. We used the common fluorescent GFP and YFP proteins as epitopes, which provide the advantage of being detectable without the use of antibodies. However, these tags can only be added to the N- and C-termini, which are both intracellular in Na_v1.5, and which are thus, not easily accessible. Therefore, we additionally chose the FLAG-epitope (Sigma-Aldrich), which consists of a short sequence that can be inserted into the extracellular loops of Na_v1.5. The results of these studies will have to be taken into account when planning the generation of a mouse line bearing an epitope-tagged Na_v1.5 channel.

Methods

Results

Electrophysiological properties of human and mouse Na_v1.5 are comparable

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).

Figure 1. Representative I_Na recordings following the current voltage (I/V)-protocol described in the Methods.

(A) Voltage-dependent currents measured for hNa_v1.5 expressed in a HEK293 cell. (B) Data from the same protocol for mNa_v1.5.

Figure 2. Electrophysiological properties of human and mouse Na_v1.5.

The voltage-clamp protocols used are shown in the corresponding insets. For B–D, the voltage x was adjusted to the voltage that elicited maximum current during the current voltage (I/V)-protocol (-10 to -30 mV). (A) I/V-protocol for assessment of reversal potentials. Peak currents were measured for both channels at -15 mV. Calculated reversal potentials are marked with square data points. (B) Voltage-dependence of activation and steady-state inactivation (SSI). The data was fitted with the Boltzmann formula. Only the slope of the inactivation curve differs between mouse and human sodium channels (shallower in mNa_v1.5). (C) Recovery from inactivation. The duration between the depolarising steps was varied from 0.25 to 3000 ms. mNa_v1.5 had a slight tendency to recover faster than hNa_v1.5. (D) Onset of slow inactivation. The duration of the first step was varied from 0.25 to 3000 ms. The relative number of channels entering slow inactivation is similar for both types. (A–B) n(hNa_v1.5) = 22, n(mNa_v1.5) = 17. (C–D) n(hNa_v1.5) = 9, n(mNa_v1.5) = 7. **P<0.01 obtained by two-tailed Student's t-tests; error bars indicate standard errors.

Table 1. Summarized properties of human and mouse Na_v1.5.

Data was obtained with current voltage (I/V)- and steady-state inactivation protocols. Mean values and standard errors are shown. **P<0.01 obtained by two-tailed Student's t-tests.

		hNav1.5	mNav1.5
		mean ± sem	mean ± sem
I/V	Vrev (mV)	51.0 ± 0.9	**56.6 ± 0.8
Activation	V1/2 (mV)	-27.8 ± 0.5	-28.2 ± 0.7
	Slope (mV/e-fold)	6.00 ± 0.17	6.24 ± 0.16
Inactivation	V1/2 (mV)	-70.0 ± 1.0	-71.7 ± 1.1
	Slope (mV/e-fold)	5.95 ± 0.12	**6.67 ± 0.19
Cell capacitance	pF	16.0 ± 1.2	14.2 ± 0.7
Imax	pA/pF	-185 ± 21	-249 ± 22
n		22	17

FLAG-tag inserted at the L299/V300 site alters voltage-dependent activation of hNa_v1.5

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.

Figure 3. Representative sodium current (I_Na) recordings.

(A) Voltage-dependent currents measured for hNa_v1.5 expressed in a HEK293 cell. The same data for (B) FLAG-hNa_v1.5 (C) YFP-hNa_v1.5, and (D) GFP-hNa_v1.5.

Table 2. Summarized properties of wild-type and tagged hNa_v1.5.

Data was obtained with current-voltage (I/V)-, and steady-state inactivation protocols. Mean values and standard errors are shown. *P<0.05, **P<0.01 obtained by two-tailed Student's t-tests (all statistics were calculated with untagged hNa_v1.5 channel as a reference).

		hNav1.5	FLAG-hNav1.5	YFP-hNav1.5	GFP-hNav1.5
		mean ± sem	mean ± sem	mean ± sem	mean ± sem
I/V	Vrev (mV)	51.8 ± 0.9	**39.3 ± 2.2	49.1 ± 1.5	53.8 ± 1.9
Activation	V1/2 (mV)	-28.9 ± 0.6	**-23.9 ± 0.5	-27.5 ± 1.1	-29.8 ± 1.1
	Slope (mV/e-fold)	5.91 ± 0.17	**6.96 ± 0.14	*6.87 ± 0.27	5.40 ± 0.36
Inactivation	V1/2 (mV)	-70.6 ± 1.2	-70.0 ± 1.1	-71.2 ± 1.4	-68.9 ± 1.2
	Slope (mV/e-fold)	5.95 ± 0.24	5.33 ± 0.15	5.69 ± 0.19	6.37 ± 0.19
Cell capacitance	pF	14.4 ± 1.6	16.3 ± 1.1	14.5 ± 0.9	14.0 ± 0.8
Imax	pA/pF	240 ± 36	**57 ± 11	*120 ± 17	214 ± 33
n		7	11	11	8

Figure 4. Electrophysiological properties of untagged and tagged hNa_v1.5.

The voltage-clamp protocols used are shown in the corresponding insets. For B–D, the voltage x was adjusted to the voltage that elicited maximum current during the current voltage (I/V)-protocol. (A) I/V-protocol for assessment of reversal potentials. Tagging with N-terminal YFP and FLAG (L299/V300) significantly decreases peak currents. Calculated reversal potentials are marked with square data points. (B) Voltage-dependence of activation and steady-state inactivation. The data was fitted with the Boltzmann formula. The activation slope of FLAG- and YFP-tagged channels is shallower compared to the untagged hNa_v1.5. V_1/2 is shifted by 5 mV for FLAG-hNa_v1.5. (C) Recovery from inactivation. The duration between the depolarising steps was varied from 0.25 to 3000 ms. No differences between the different channels could be detected. (D) Onset of slow inactivation. The duration of the first step was varied from 0.25 to 3000 ms. The relative number of channels entering slow inactivation is similar for all four channel types. (A–B): n(untagged) = 7, n(FLAG) = 11, n(YFP) = 11, n(GFP) = 8. (C): n(untagged) = 12, n(FLAG) = 5, n(YFP) = 11, n(GFP) = 8. (D): n(untagged) = 10, n(FLAG) = 8, n(YFP) = 10, n(GFP) = 8. **P<0.01 obtained by two-tailed Student's t-tests; error bars indicate standard errors.

Discussion

The present study demonstrates (1) that the biophysical properties of mouse Na_v1.5 are essentially similar to the human homolog when expressed in HEK293 cells, and (2) that adding epitopes either upstream of the N-terminus of human Na_v1.5 or in one of the extracellular loops reduces the amount of I_Na and alters some of its biophysical properties. Interestingly, GFP in the N-terminus was the only epitope that did not modify any of the measured biophysical properties of hNa_v1.5. The most pronounced effects could be observed by the insertion of the FLAG-tag in an extracellular loop. In this construct, not only was the amount of I_Na drastically decreased, but also the activation properties of the sodium channel were altered. The smaller changes found in the properties of YFP-hNa_v1.5 might be partially linked to the different vector used for this epitope, especially since no alterations could be observed for GFP-hNa_v1.5.

The limitations of studying mutant Na_v1.5 channels in mammalian cells have been demonstrated in two recent studies. First, Mohler and colleagues⁸ observed that the Brugada syndrome causing mutant p.E1053K Na_v1.5 channel did not display any trafficking defect in HEK293 cells, while it failed to traffic to the intercalated discs when expressed in rat ventricular cells. Second, the Na_v1.5 p.D1275N variant, found in patients with dilated cardiomyopathy and various arrhythmias and conduction disease, was also found to display reduced expression in knocked-in mouse cardiac tissue and defective expression at the lateral membrane of ventricular myocytes⁹. However, when expressed in chinese ovary cells, the p.D1275N variant had properties that were undistinguishable from wt channels. These observations demonstrate that, while useful to study their intrinsic biophysical properties, the mammalian cells that are used as expression systems have clear limitations when studying the trafficking properties of ion channels. Generation of genetically-modified animal models is one of the most powerful, albeit time-consuming, approaches.

However the findings of the present study have to be taken into account when planning to generate such mouse models that harbour specific epitopes in the mouse Na_v1.5 gene. Different combinations of epitopes and insertion sites might reveal better candidates for in-vivo approaches. Furthermore, additional studies should be performed in HEK293 cells co-expressing other subunits and regulating proteins, and in native cardiomyocytes in order to assess the effects of added epitopes on the interactions with these proteins.