SCN5A mutation G615E results in NaV1.5 voltage-gated sodium channels with normal voltage-dependent function yet loss of mechanosensitivity

ABSTRACT SCN5A is expressed in cardiomyocytes and gastrointestinal (GI) smooth muscle cells (SMCs) as the voltage-gated mechanosensitive sodium channel NaV1.5. The influx of Na+ through NaV1.5 produces a fast depolarization in membrane potential, indispensable for electrical excitability in cardiomyocytes and important for electrical slow waves in GI smooth muscle. As such, abnormal NaV1.5 voltage gating or mechanosensitivity may result in channelopathies. SCN5A mutation G615E – found separately in cases of acquired long-QT syndrome, sudden cardiac death, and irritable bowel syndrome – has a relatively minor effect on NaV1.5 voltage gating. The aim of this study was to test whether G615E impacts mechanosensitivity. Mechanosensitivity of wild-type (WT) or G615E-NaV1.5 in HEK-293 cells was examined by shear stress on voltage- or current-clamped whole cells or pressure on macroscopic patches. Unlike WT, voltage-clamped G615E-NaV1.5 showed a loss in shear- and pressure-sensitivity of peak current yet a normal leftward shift in the voltage-dependence of activation. In current-clamp, shear stress led to a significant increase in firing spike frequency with a decrease in firing threshold for WT but not G615E-NaV1.5. Our results show that the G615E mutation leads to functionally abnormal NaV1.5 channels, which cause disruptions in mechanosensitivity and mechano-electrical feedback and suggest a potential contribution to smooth muscle pathophysiology.

Mechanosensitivity is important for the function of all cells [7], but it plays an especially important role in organ systems whose primary function is mechanical, such as cardiovascular, gastrointestinal, and urinary. In electrically excitable systems, mechanical forces regulate function by mechanoelectrical feedback [8]. For example, mechanical stretch of neurons reversibly depolarizes the resting membrane potential and increases the frequency of action potentials [9], which is balanced by mechanosensitive voltage-gated potassium channels that provide a "mechanical brake" to their excitability [10]. Na V 1.5 channels are gated by voltage, but they are also mechanosensitive [11,12], and mechanosensitivity of these channels is particularly relevant because they are expressed in heart and gut, which are mechanically active organs. Indeed, disruptions in Na V 1.5 mechanosensitivity may contribute to cardiac conduction disorders [13]. In cardiomyocytes (7), GI SMCs [14,15], and heterologous expression systems [11,12,16], mechanical stimuli alter Na V 1.5 function by increasing peak Na + current (I PEAK ), hyperpolarizing the voltage dependence of activation (V 1/2A ) and availability (inactivation, V 1/2I ), and accelerating channel kinetics. However, the mechanism of Na V 1.5 mechanosensitivity remains unclear, which limits our ability to understand the contributions of Na V 1.5 mutations to pathophysiology.
Previous work showed that disease-associated Na V 1.5 mutations can disrupt voltage-gating, and a portion also disrupts mechanosensitivity [5,6]. Testing whether any Na V 1.5 mutation could affect these two functions separately may illuminate the relationship between these functions. The mutation G615E Na V 1.5 was found in several studies to associate with cardiac conduction disorders [17][18][19][20] and irritable bowel syndrome [4] but does not appear to lead to significant disruptions in Na V 1.5 voltagedependent function. In this study, we compare the mechanosensitivities of wild-type (WT) and G615E Na V 1.5, a missense mutation in the DI-DII linker with normal current density [4] but a potentially disrupted mechanosensitivity [21].

Molecular biology
Plasmids A single nucleotide change (c.1844 G→A) was engineered by site-directed mutagenesis in a construct containing the most common splice variant of SCN5A (hH1c1, H558/Q1077del) to substitute G615E in the Na + channel α-subunit (p.G615E-Na V 1.5) using the QuikChange II XL Site-Directed Mutagenesis Kit. The integrity of the construct and the presence of the desired mutation were verified by DNA sequencing.

Electrophysiology
Pipette fabrication and data acquisition Electrodes were pulled to a resistance of 2-5 MΩ from KG12 (Kimble glass, Fisher Scientific, Massachusetts, USA) for whole-cell voltage-or current-clamp or to a resistance of 1-2 MΩ from Garner 8250 glass for cell-attached pressure-clamp on a P97 puller (Sutter Instruments, California, USA) and coated with heat-cured R6101 polymer (Dow Corning, MI). Whole-cell and cell-attached patch data from HEK-293 cells were recorded at 20 kHz with an Axopatch 200B patch clamp, CyberAmp320, Digidata 1550, and pClamp 10.5 software (Molecular Devices, California, USA).

Whole-cell voltage clamp
The intracellular solution contained (in mM): 135 K + , 130 CH 3 SO 3 − , 20 Cl − , 5 Na + , 5 Mg 2+ , 5 HEPES, 2 EGTA; pH 7.0, 290 mmol/kg. The extracellular solution contained (in mM): 15 Na + , 140 Cs + , 160 Cl − , 2.5 Ca 2+ , 5 K + , 10 HEPES, 5.5 glucose; pH 7.35, 305 mmol/kg. Episodic protocol. To measure peak Na + current density, cells transfected with WT-or G615E-Na V 1.5 were held at −120 mV before pulsed through a 2-stage, 24-step voltage ladder (1) from −80 to +35 mV in 5 mV intervals for 50 ms each and (2) to −30 mV for 50 ms. The times between sweeps and each of 10 runs were 250 ms and 6 s, respectively. Peak currents at each voltage step were normalized to the cell capacitance (pF) dialed in during recording or to the maximum peak inward current without shear. Mechanical stimulation by shear stress. Flow of extracellular (bath) solution was applied by gravity drip, calibrated to a rate of 10 mL/min with intravenous tubing.

Whole-cell current clamp
The intracellular solution contained (in mM): 135 K + , 130 CH 3 SO 3 − , 20 Cl − , 5 Na + , 5 Mg 2+ , 5 HEPES, 2 EGTA; pH 7.0, 290 mmol/kg. The extracellular solution contained (in mM): 150 Na + , 160 Cl − , 5 K + , 2.5 Ca 2+ , 10 HEPES, 5.5 glucose; pH 7.35, 305 mmol/kg. Gap-free protocol. To measure the change in frequency of spontaneous events, cells transfected with WT-or G615E-Na V 1.5 were recorded continuously below the predicted threshold. Briefly, window currents were plotted automatically from whole-cell Na + currents recorded in voltage-clamp mode. With the range of the window current calculated to determine the threshold to elicit membrane potential spikes, the amplifier was switched to I-clamp mode, and current was continuously injected to hyperpolarize the membrane potential approximately 10-20 mV negative from the window current. Spontaneous activity was recorded with a gapfree protocol. With current injected to keep the resting potential hyperpolarized relative to the half-point of the voltage-dependence of inactivation in order to ensure full availability (WT, −89.9 ± 3.8 mV vs. G615E, −91.4 ± 3.5 mV; n = 8, P = 0.49 by a two-tailed unpaired t-test), spontaneous spike frequencies without shear ranged from 0.1 to 1.0 Hz for 80-90% of all experiments (8 of 9 WT and 18 of 22 G615E); experiments with baseline frequencies outside this range were excluded from the analysis. Episodic protocols. To establish a prediction for the threshold of elicited activity, cells transfected with WT-or G615E-Na V 1.5 were held at −15 pA before pulsed through a 9-step current ladder from −15 pA to +25 pA in 5 pA intervals for 50 ms each. The time between sweeps was 1 s. To measure the probability of firing potentials, cells were held at resting current (e.g., −15 pA) and pulsed through 20 repetitions of a five-stage protocol with 5.45 s between sweeps: (1) to above the predicted threshold (e.g., +0 pA) for 50 ms and back to rest for 500 ms, then (2)(3)(4)(5) to below the predicted threshold (e.g., −5 pA) for 50 ms and back to rest for 500 ms. Mechanical stimulation. Shear stress was applied by flow of extracellular solution at 10 mL/min, as described above in voltage-clamp mode.

Data analysis
To calculate whole-cell conductance and voltage dependence of activation, Na V 1.5 current-voltage (I-V) plots were fit with the equation: I V = G MAX * (V-E REV )/(1 + e (V-V1/2A)/slope ), in which G MAX is the maximum conductance of peak Na + current, and V 1/2A is the voltage of half-activation. To calculate frequency, the number of spontaneous potentials firing past 0 mV in gap-free mode were expressed as a fraction of the 60-to 120-s acquisition time (Hz). To calculate the probability of firing, the number of evoked potentials firing past 0 mV were expressed as a fraction of the number of current stimuli. Response to pressure was measured by the change in peak Na + current, I STEP2 -I STEP1 = ΔI PEAK . I-V curves were examined for any shifts in the V 1/2 of activation (ΔV 1/2A ) versus paired controls without applied pressure. Data are expressed as the mean ± standard error of the mean (SEM). Change as a result of shear stress or pressure was assigned when P < 0.05 for mechanostimulus to control by a one-sample t-test ( Figure 1 (f,h); Figure 2(g-i); Figure 4(d); Figure 5(i)), P < 0.05 for G615E to WT by a two-way ANOVA with Dunnett post-test ( Figure 3(e-f), Figure 4(c)), or P < 0.05 for G615E to WT by a three-way ANOVA with Tukey post-test ( Figure 1(b,d); Figure 2(a-f); Figure 3(g-h); Figure 5(e-h)).
Effect of pressure on WT and G615E Na V 1.5 current within a patch Next, we used another technique to confirm the loss of G615E Na V 1.5 mechanosensitivity we found with shear stress. We examined WT and G615E Na V 1.5 by simultaneous voltage-and pressure-clamp with on-cell patches [16,22]. With a two-step protocol to determine Na V 1.5 pressure dependence [22] ( Figure  3(a-b)), the currents elicited by "Voltage Step 1" test the voltage-dependence of Na V 1.5, while currents from "Voltage Step 2" test the effect of pressure concurrently with voltage. We found that during Voltage Step 1 (0 mmHg), peak Na + currents and voltage-dependence of activation were not statistically different between constructs (I MAX : WT, 71.6 ± 15.0 pA vs. G615E, 39.0 ± 12.8 pA, n = 9, P = 0.12; V 1/2A : WT, 41.2 ± 4.5 mV vs. G615E, 39.4 ± 1.7 mV, n = 9, P = 0.71 WT to G615E by two-tailed unpaired t-tests). However, as with shear stress, there were significant differences in the responses of WT and G615E Na V 1.5 to pressure (Table 1, Figure 3(c-f)). inactivation components (h-i) of WT (black) or G615E (red) Na V 1.5 currents at −30 mV (n = 12 cells each; *P < 0.05% to 0% by a twotailed one-sample t-test; †P < 0.05 to WT by a two-tailed unpaired t-test).  WT Na V 1.5 V 1/2A was hyperpolarized proportionately with pressure, for a slope of −1.0 ± 0.1 mV per −10 mmHg (P < 0.05 effect of pressure by a three-way ANOVA with Tukey post-test) (Figure 3 (e,g)), and WT Na V 1.5 macroscopic peak currents increased proportionately with negative patch pressure, for a rate of 3.9 ± 0.7% per −10 mmHg (n = 10 cells, P < 0.05 effect of pressure, voltage, and interaction by a two-way ANOVA with Dunnett posttest) (Figure 3(e,h)). G615E Na V 1.5 V 1/2A left-shifted with pressure (P < 0.05 effect of pressure and P > 0.05 effect of genotype) (Figure 3(f-g)), and the slope of ΔV 1/2A for G615E Na V 1.5 did not differ from WT Na V 1.5, −0.9 ± 0.3 mV per −10 mmHg (Figure 3(g), Table 1). In contrast to WT Na V 1.5, peak Na + currents of G615E did not respond to pressure, for a rate of only 0.2 ± 1.0% per −10 mmHg (n = 10 cells, P < 0.05 effect of genotype, P < 0.05 effect of interaction between genotype and pressure by a three-way ANOVA with Tukey post-test) (Figure 3(h), Table  1). In all, our data in both whole cell with shear stress and patch with pressure show that G615E Na V 1.5, unlike WT Na V 1.5, lacks substantial forcedependent changes in mechanically induced peak currents and kinetics, whilst retaining the responses in voltage-dependence.

Effect of shear stress on cell electrical excitability
Since Na V 1.5 is involved in electrical excitability in the heart and gut, we pursued the effects of mechanical stimulation on Na V 1.5 function in current clamp. Recent studies show that electrical excitability can be re-created in mammalian cell lines commonly used for heterologous expression, such as CHO and HEK-293 cells (14,15). Therefore, we examined the spontaneous spiking of WT or G615E-transfected Na V 1.5 HEK-293 cells for changes during shear stress in currentclamp mode (Figure 4(a-b)). For WT Na V 1.5, we saw that shear stress reversibly increased the probability of spontaneous spiking events (Figure 4  (a-b)); however, for G615E Na V 1.5, shear stress failed to produce an increase in spiking frequency a b c d WT shear and P < 0.05 interaction between stimulus, genotype, and shear by a three-way ANOVA with Tukey post-test). (g-h), Time to peak potential (t PEAK ) from cells expressing WT (g) or G615E-Na V 1.5 (h) channels evoked at the 15-pA (■) or 10-pA (□) stimulus before (CTRL) or during shear stress (SHEAR) (n = 6 cells each; *P < 0.05, WT control vs. WT shear; P < 0.05, effect of stimulus or effect of shear; and P < 0.05, interaction between genotype and shear by a three-way ANOVA with Tukey post-test). (i), Average change in time to peak potential (Δt PEAK ) induced by shear for WT (black) or G615E-Na V 1.5 (red) (n = 6 cells each; *P < 0.05% to 0% by a two-tailed one-sample t-test).
We next wanted to test whether elicited excitability is different for WT and G615E Na V 1.5 channels. In current clamp mode, a -15-pA injection for 50 ms elicited a singular membrane potential spike in HEK-293 cells transfected with either WT or G615E Na V 1.5 ( Figure 5(a-b)). However, the shear-induced changes to the membrane potential spiking kinetics of WT Na V 1.5 were not observed in cells with G615E Na V 1.5 ( Figure 5(b)). Since our data suggested an acceleration in Na V 1.5 activation and inactivation with mechanical stress, we wanted to test whether submaximal electrical stimulation would result in increased electrical excitability in the presence of mechanical stimulation. Therefore, we designed a protocol that compared a step at maximal (fully activating) stimulation to a sequence of four steps at submaximal stimulation ( Figure 5(c-d), top traces). At rest, we saw that at maximal stimulation (15 pA), >90% of steps led to spiking for both WT (98.3 ± 1.7%, n = 6, Figure 5(c,e)) and G615E Na V 1.5 (90.8% ±7.2%, n = 6, P> 0.05 vs. WT by a two-tailed nonparametric t-test; Figure 5(d,f)). Meanwhile, submaximal stimulation (10 pA) at rest led to spiking in a similar but reduced fraction of stimuli for both WT and G615E (WT, 23.4 ± 9.1% vs. G615E, 17.3 ± 4.6%; n = 6 each, P> 0.05 by a two-tailed nonparametric t-test). In the presence of shear stress, maximal stimulation continued to produce membrane potential spikes for >90% of depolarizations for both WT and G615E Na V 1.5 (WT, 92.5 ± 4.8%; G615E, 92.4 ± 5.9%; n = 6 each, P > 0.05 by a twotailed non-parametric t-test; Figure 5(c-f)), but only in WT and not in G615E did shear stress increase potentials induced by subthreshold stimuli (WT, 71.9 ± 14.4%; G615E 27.2 ± 8.5%; P < 0.05 WT control vs. WT shear and P < 0.05 interaction between stimulus, genotype, and shear by a threeway ANOVA with Tukey post-test).

Discussion
The goal of the current study was to examine Na V 1.5 mechanosensitivity and its role in cellular mechano-electrical feedback. We used a unique SCN5A mutation G615E that is associated with acquired long-QT syndrome [19], sudden cardiac death [17], and irritable bowel syndrome [4]. In previous studies [4,17] and in this one, G615E Na V 1.5 had mostly intact voltage-dependent functionnormal voltage-dependence of activation, and either no or minor changes in voltagedependence of inactivation.
On the other hand, we found dramatic differences in mechanosensitivity of voltage-dependent gating between WT and G615E Na V 1.5. We tested both constructs in whole cell and patch, using shear stress and patch pressure as mechanical stimuli, respectively. For WT Na V 1.5, we saw force produce several changes to voltage-dependent function, similar to previous studies on Na V 1.5 [5,11,12,16,22] and Na V 1.4 [23], but these changes were nearly absent for G615E Na V 1.5. Thus, G615E Na V 1.5 joins previously identified diseaseassociated mutations with abnormal Na V 1.5 mechanosensitivity. These associated with long QT syndrome [13] in the heart and with IBS [5,6] in the gut and resulted in Na V 1.5 with abnormalities in voltage-dependent function that were further accentuated by mechanical forces. However, to our knowledge, this is the first Na V 1.5 mutation that has disrupted mechanosensitivity, while voltage-sensitivity remained mostly intact. Our findings may be relevant for understanding channelopathy mechanisms in cases when Na V 1.5 mutations fail to reveal functional changes using voltage-dependence protocols. In such cases, and as we see with G615E Na V 1.5, the functional impact may be on mechanical [6] or thermal [24] sensitivity.
Our results provide intriguing mechanistic insights on Na V 1.5 mechanosensitivity. First, G615E is located on the intracellular linker connecting DI and DII, which is a novel location for a missense mutation to impact mechanosensitivity. Other Na V 1.5 channelopathies like G298S have loss-of-mechanosensitivity and are in linkers as well. However, how these linkers contribute to the mechanism of Na V 1.5 mechanosensitivity remains unclear. Second, our findings suggest that mechanisms of voltage-and mechano-sensation by Na V 1.5 may be distinct. This is surprising since mechanical stimuli are well established to modulate voltage-sensitivity of voltage-gated channels [11,12,25] but not to introduce a separate mechanogating paradigm [11,12]. Third, G615E Na V 1.5 lost one but not both mechanosensitive responsesit lacked a perfusion-induced current increase but maintained a negative shift in the voltage-dependence of activation. This would suggest that mechanosensitive increases in peak Na + current may be mechanistically distinct from mechanically induced shifts in voltagedependence of activation and availability. Fourth, the findings suggest that the mechanosensitivity of the voltage-dependence of activation may be separate from that of availability. Previous and current studies show that mechanical stimuli accelerate the kinetics of activation and inactivation by the same constant. If the acceleration of activation by force is the rate-limiting step [26], it may explain the effect on the acceleration of inactivation. However, G615E Na V 1.5 demonstrates an intact mechanosensitivity of activation but a loss of mechanosensitivity of inactivation, suggesting separate mechanisms. In all, our results shed important light on the mechanism of Na V 1.5 mechanosensitivity and show that Na V 1.5 mechano-and voltagesensitivity may be targeted separately.
We are ultimately interested in understanding how Na V 1.5 mechanosensitivity impacts SMC excitability, also called mechano-electrical feedback [8]. These cells undergo constant repetitive mechanical deformationsthey are stretched during diastole and contracted during systole. Stretch is excitatory for WT Na V 1.5 channels at the upstroke. But given the acceleration of inactivation, Na V 1.5 stretch also results in a more significant current decrease during inactivation [11][12][13] and slowed recovery from inactivation [11]. Therefore, the overall impact of Na V 1.5 mechanosensitivity on SMC function is difficult to judge only from the voltage-dependent operation. We designed current-clamp protocols in a reductionist system, a HEK-293 cell that expressed only WT Na V 1.5 or G615E Na V 1.5. Similar to previous studies [27,28], we found that these cells had both spontaneous and elicited electrical excitability. Compared to WT, G615E Na V 1.5 had decreased mechanosensitivity of both spontaneous and elicited firing, suggesting that Na V 1.5 mechanosensitivity may play an important excitatory role in SMC mechano-electrical feedback. However, a note of caution is required. We set the resting potential hyperpolarized to allow for full Na V 1.5 availability, but this system limits our ability to determine whether sub-threshold events by either channel can result in firing, as may happen in excitable cells. In a set of preliminary studies, we investigated the potential influence of G615E-Na V 1.5 on mechano-electrical feedback in GI SMCs by computational modeling. In silico modeling simulated Na V 1.5 voltage-and currentclamp in vitro behavior, and the resulting SMC electrical activity and cytoplasmic Ca 2+ concentrations were affected by mechanical forces for WT but not G615E-Na V 1.5 (Supplementary Figure 2).
In addition to mechanosensitive voltage-gated sodium channels, cells have other mechanosensitive voltage-gated ion channels, such as potassium (K V ) [29] and mechanically gated ion channels [30]. The effects of mechanical forces on these channels are expected to produce various electrical outcomes. For example, K V 1.1 mechanosensitivity leads to "mechanical braking" of neuronal excitability [10]. Further, it will be important to determine the precise mechanical energies that the mechanosensitive ion channels encounter in vivo and to replicate these for studies in vitro. It is unclear how the current in vitro mechanostimulation protocols correlate with in vivo physiology. To fully understand mechano-electrical coupling we will need to integrate mechanosensitivity of Na V 1.5 and other mechanosensitive ion channels into cell models and to place these models into physiologically relevant mechanical contexts.
In summary, the disease-associated Na V 1.5 missense mutation G615E disrupts Na V 1.5 mechanosensitivity without a significant impact on voltage-dependent function, which may have important consequences for mechano-electrical coupling in myocytes. This raises the possibility of directly targeting Na V 1.5 mechanosensitivity in disease with a drug such as ranolazine [16,22], which does not have a significant effect on Na V 1.5 peak current but can inhibit mechanosensitivity [15,16,31].