Cationic Modulation of Voltage-Gated Sodium Channel (Nav1.5): Neonatal Versus Adult Splice Variants—1. Monovalent (H⁺) Ions

Abstract

Background: Voltage-gated sodium channels are functionally expressed in human carcinomas. In breast and colon cancers, the neonatal splice variant of Nav1.5 (nNav1.5) is dominant. This differs from the adult (aNav1.5) by several amino acids, including an outer charge reversal (residue-211): negatively charged aspartate (aNav1.5) versus positively charged lysine (nNav1.5). Thus, nNav1.5 and aNav1.5 may respond to extracellular charges differently.

Materials and Methods: We used whole-cell patch-clamp recording to compare the electrophysiological effects of the monovalent cation hydrogen (H⁺) on nNav1.5 and aNav1.5 expressed stably in EBNA cells.

Results: Increasing the H⁺ concentration (acidifying pH) reduced channel conductance and inhibited peak currents. Also, there was a positive shift in the voltage dependence of activation. These changes were significantly smaller for nNav1.5, compared with aNav1.5.

Conclusions: nNav1.5 was more resistant to the suppressive effects of acidification compared with aNav1.5. Thus, nNav1.5 may have an advantage in promoting metastasis from the acidified tumor microenvironment.

Introduction

Voltage-gated sodium channels (VGSCs) open upon membrane depolarization, generating the transient Na⁺ current (I_Na) responsible for membrane “excitability” and propagation of action potentials.^1,2 Nav1.5 is the major VGSC α-subunit (VGSCα) expressed normally in cardiac muscle, and the activation/inactivation of this channel is vital for normal heart rhythm.³ Consequently, slight changes in Nav1.5 functioning have been associated with a wide range of cardiac conduction disorders, including Brugada syndrome, long-QT syndrome, idiopathic ventricular fibrillation, and arrhythmias.^3,4

Alternative splicing occurs in VGSCα genes.⁵ A major splicing event in SCN5A (the gene encoding Nav1.5) involves exon-6 encoding the region of the voltage sensor/paddle of domain I (DI), including segment 3 (S3), most of segment 4 (S4), and the S3–S4 extracellular linker.^6,7 Such DI:S3/S4 splicing was originally described for Nav1.2 and Nav1.3 in rat brain and was shown to be developmentally regulated; transcripts possessing the 5′-exon occur at birth but are rapidly replaced by the 3′-exon.^8,9 Accordingly, the 5′ and 3′ variants are referred to as “neonatal” and “adult”, respectively. For Nav1.5, the exon-6 variant proteins differ by seven amino acids, including a “conserved” negatively charged aspartate at position 211 in the adult (aNav1.5) being replaced by a positively charged lysine in the neonatal (nNav1.5).⁶

In neonatal (1-day old) mouse heart, nNav1.5 protein expression occurs at a level comparable with aNav1.5 but cannot be detected in adult tissue.⁶ Re-expression of nNav1.5 can occur in mature cardiomyocytes under pathophysiological conditions (e.g., cardiac hypertrophy), where there is a shift to “fetal” ion channels phenotypes.^10,11 Interestingly, nNav1.5 is functionally expressed de novo in human metastatic breast and colon cancer cells, where it potentiates Matrigel invasion in vitro as well as metastasis in vivo.^12–19 nNav1.5 expression has also been described in human neuroblastoma cells.²⁰

Previously, we demonstrated that aNav1.5 and nNav1.5 expressed stably in EBNA-293 cells differ in their electrophysiological profiles.²¹ In particular, nNav1.5 channels exhibited significantly more depolarized voltage of activation, hyperpolarized inactivation, slower recovery from inactivation, and slower activation and inactivation kinetics. Changing Lys²¹¹ back to Asp²¹¹ in nNav1.5 abolished most of the differences, suggesting that the positive electric field induced by Lys²¹¹ in the voltage-sensing region of DI was primarily responsible for the observed differences.²¹ Such change of local electrostatic potential could also affect the interaction with cations and impact channel functioning, especially since the charge reversal is near the voltage sensor. Cations generally are known to inhibit I_Na in at least two ways: (1) reduction of maximal Na⁺ conductance and (2) depolarization of voltage dependence of gating.^1,22,23 These effects are thought to involve, respectively, pore blockage and neutralization of negative charges on protein surface, for example, external sialic acids and carboxylates.^1,22 Thus, nNav1.5 versus aNav1.5 may respond to extracellular charges differently. Effects of various cations on I_Na were studied in native cardiomyocytes,^24,25 and cardiac Purkinje cells.^22,26 However, the contributing VGSC(s) were not clear since VGSCαs isoforms additional to Nav1.5 would be present, including Nav1.1–1.4 and Nav1.6.^27,28 In contrast, there is less work on cationic modulation of Nav1.5 in isolation.^29–31 Furthermore, it is unclear whether the Nav1.5 DI:S3/S4 splice variants would respond differently to cations. It is our hypothesis that the Asp²¹¹ to Lys charge reversal would result in the effects of cations on channel gating being smaller for nNav1.5 versus aNav1.5.

In this study, we determined the comparative electrophysiological effects of the monovalent cation hydrogen (H⁺) on nNav1.5 versus aNav1.5. H⁺ was selected also because acidosis occurs under hypoxia, including that experienced by ischemic cardiomyocytes and growing tumors.^32,33 Indeed, the in vivo tumor microenvironment is well known to be acidic (pH ∼6.5), compared with the normal body pH of ∼7.4. The acidity promotes the invasiveness of cancer cells through a variety of mechanisms, including activation of proteolytic enzymes.³³ Results of studies on divalent and trivalent cations are presented in the accompanying article.³⁴

Materials and Methods

Cell culture

Production of EBNA-293 cell lines stably expressing recombinant aNav1.5 and nNav1.5 was described previously.⁶ Cells were grown as described.²¹ Cells were maintained in 100 mm Falcon tissue culture dishes (Becton Dickinson) at 37°C, 5% CO₂ and 100% relative humidity. For electrophysiology, cells were plated into 35 mm dishes at 10–30% confluence and used within 48 h.

Electrophysiology

Whole-cell patch-clamp recordings were performed as described.²¹ Patch pipettes were made from borosilicate glass (GC100-F; Harvard Apparatus) using a two-stage puller (PP830; Narashige) and heat polished to 3–5 MΩ when filled with a pipette solution, as described.²¹ Cs⁺ was included in the internal patch solution to block endogenous K⁺ currents.³⁵ Membrane currents were recorded using Axopatch 1D (Axon Instruments) at room temperature, as described.²¹ Capacitative transients were cancelled and series resistance (R_s) errors were compensated by >85% with a lag of 8 μs. To limit the voltage drop across R_s to <5 mV, data were accepted only as follows: (1) Isolated round cells (≤20 pF) were used. (2) Cells expressed peak I_Na <6 nA. (3) Activation slope factors were >4.5 mV; steeper slopes would indicate voltage-clamp loss.^36,37 (4) Seal resistances were >1 GΩ and initial uncorrected R_s (<10 MΩ) changed <20% during recording. (5) Cells with I_Na < 500 pA were discarded to avoid bias from any endogenous I_Na.³⁵

Solutions

Before recording, growth medium was replaced by perfusing cells for ∼15 min with mammalian physiological saline (MPS), as described.²¹ For changing the H⁺ level, MPS was titrated with NaOH or HCl to the desired pH (this was confirmed to have remained within 10% of the set value after the recordings). For pH <6.5, HEPES was replaced with MES buffer. The buffer change itself at control pH (7.25) had no effect on I_Na (data not shown). The solutions were bath-applied at a rate of 1.5 mL/min using a gravity-fed perfusion system. Recordings started 4–5 min after seal rupture to allow time for stabilization.³⁸ In each recording, cells were exposed to “control” and “test” MPS solutions alternatively for at least 2 min each (perfusion switch time <2 s) and voltage pulses were applied in each condition. In all experiments, the order of applying the various doses was randomized. Data were accepted only if the pre- and post-measurements were <10%. Chemicals were from Sigma.

Voltage pulse protocols

Four main types of voltage pulse protocols were used, described previously,²¹ as follows: (1) Dose–response, (2) current–voltage (I–V), (3) steady-state inactivation, and (4) recovery from inactivation.

Curve fitting and data analysis

Conductance–voltage (G–V) relationships were determined using the equation: G = I/(V − V_rev), where G is conductance, I is current amplitude, V is test voltage, and V_rev is the reversal potential for Na⁺. For each cell, normalized conductance and steady-state availability were plotted as a function of membrane potential, and fit with a Boltzmann function of the form:

where G_max is maximal conductance, V_m is membrane voltage, V_1/2 is voltage at which the current is half activated/inactivated, and k is slope factor of voltage sensitivity.

The voltage dependence of time to peak and the time course of inactivation (i.e., decay of current from peak) were fit to the following single and double exponential decay functions, respectively:

where A₁ and A₂ are the coefficients of decline of the time constant (τ) with voltage, V is membrane potential and V₀ is a constant describing the voltage dependence of τ. For the double-exponential equation, A_s and A_f are the maximal amplitudes of the slow and fast components of the total current; τ_fast and τ_slow are the time constants for decay of the fast and slow components of the current, respectively; the constant C is the asymptote. For the time course of recovery from inactivation, I_t/I_c was plotted as a function of recovery time (Δt) and fitted to the single exponential function.

Values of G_max at various H⁺ concentrations were normalized relative to control values. Dose–response curves for G_max, activation V_1/2, T_peak, and τ_fast were fit with the following modified version of the logistic equation since the concentration axis was logarithmic:

where pK_a is the pH at which half-maximum effect occurred.³⁸ All dose–response relationships produced fits where n was not statistically different from unity. It should also be noted that in the extracellular pH (pH_o) range tested V_1/2 shifts did not saturate, which may have precluded accurate estimation of pK_a.

Data analyses

Data were obtained from a minimum of three separate independently plated dishes per condition and combined to give an overall mean ± standard error. Each parameter was determined from a minimum of 10 cells. Statistical analyses were performed using Microsoft Excel and Origin 6.1. Statistical significance was determined with paired or unpaired Student's t-test, as appropriate. Results were considered significant for p < 0.05 (*) and highly significant for p < 0.01 (**).

Results

Effects of H⁺ on the electrophysiological parameters were studied in the pH range 8.25 to 5.25 changed in steps of 0.5. For all the conditions and parameters studied, the average data obtained for the effects of extracellular H⁺ (pH_o) on nNav1.5 and aNav1.5 are given and compared in Tables 1 and 2.

Table 1.

Effects of H⁺ on Neonatal and Adult Nav1.5 Electrophysiological Parameters

Parameter	Splice variant	Control	pH 8.25	pHo 6.75	pHo 6.25	pHo 5.75	pHo 5.25	Recovery
Relative Gmax (%)	nNav1.5	100.0 ± 0.0	99.3 ± 5.6	87.8 ± 2.5	69.5 ± 3.2	45.3 ± 2.6	21.4 ± 1.3	99.3 ± 4.2
	aNav1.5	100.0 ± 0.0	100.2 ± 3.7	85.9 ± 2.8	70.3 ± 1.5	42.7 ± 1.4	20.2 ± 1.5	104.9 ± 2.3
Vthres (mV)	nNav1.5	−55.4 ± 0.9	−57.8 ± 0.9	−53.8 ± 1.0	−49.4 ± 1.2	−43.0 ± 1.8	−33.1 ± 2.0	−58.2 ± 1.0
	aNav1.5	−60.8 ± 0.8	−62.5 ± 0.9	−59.8 ± 1.1	−55.8 ± 0.8	−47.0 ± 1.2	−35.0 ± 1.2	−62.3 ± 0.7
Vpeak (mV)	nNav1.5	−7.3 ± 1.6	−10.0 ± 1.9	−6.5 ± 2.6	−1.9 ± 1.8	5.3 ± 3.0	16.9 ± 2.5	−9.1 ± 2.0
	aNav1.5	−22.5 ± 1.9	−25 ± 1.8	−21.0 ± 2.4	−15.8 ± 2.0	−2.7 ± 1.7**	11.0 ± 2.9*	−23.6 ± 1.7
Activation V1/2 (mV)	nNav1.5	−27.3 ± 1.4	−30.7 ± 1.3	−26.0 ± 2.0	−21.7 ± 1.7	−15.1 ± 2.8	−3.3 ± 2.5	−29.6 ± 1.6
	aNav1.5	−39.2 ± 1.2	−42.1 ± 0.8	−39.3 ± 1.6	−32.3 ± 1.3	−22.9 ± 1.5**	−7.5 ± 2.0*	−41.4 ± 1.1
Activation k	nNav1.5	7.5 ± 0.4	7.4 ± 0.5	8.3 ± 0.4	8.5 ± 0.4	9.5 ± 0.4	10.0 ± 0.6	8.0 ± 0.3
	aNav1.5	5.2 ± 0.3	5.1 ± 0.4	5.2 ± 0.4	6.2 ± 0.3	7.6 ± 0.3**	10.1 ± 0.6**	5.0 ± 0.2
Inactivation V1/2 (mV)	nNav1.5	−89.0 ± 1.6	—	—	−84.3 ± 1.3	—	−74.1 ± 1.4	−92.1 ± 1.5
	aNav1.5	−91.0 ± 1.1	—	—	−86.1 ± 1.2	—	−77.1 ± 1.1	−93.0 ± 1.4
Inactivation k	nNav1.5	−6.1 ± 0.2	—	—	−6.2 ± 0.2	—	−5.8 ± 0.3	−6.4 ± 0.2
	aNav1.5	−5.8 ± 0.2	—	—	−6.0 ± 0.2	—	−6.2 ± 0.7	−6.1 ± 0.2
V0 (mV)	nNav1.5	19.8 ± 1.0	19.8 ± 1.6	24.9 ± 2.1	24.3 ± 2.1	23.1 ± 1.8	32.4 ± 9.4	21.2 ± 1.9
	aNav1.5	20.7 ± 1.6	24.2 ± 2.4	22.3 ± 1.0	20.9 ± 1.7	18.8 ± 1.1	30.4 ± 8.0	25.2 ± 1.4
Tpeak at 0 mV (ms)	nNav1.5	0.77 ± 0.03	0.71 ± 0.01	0.79 ± 0.04	0.92 ± 0.03	1.10 ± 0.07	1.44 ± 0.08	0.69 ± 0.02
	aNav1.5	0.58 ± 0.02	0.55 ± 0.01	0.61 ± 0.02	0.70 ± 0.02	0.87 ± 0.03	1.16 ± 0.04	0.57 ± 0.02
τfast at 0 mV (ms)	nNav1.5	0.91 ± 0.06	0.84 ± 0.02	0.97 ± 0.08	1.17 ± 0.10	1.42 ± 0.14	2.09 ± 0.25	0.85 ± 0.06
	aNav1.5	0.64 ± 0.02	0.61 ± 0.04	0.68 ± 0.03	0.85 ± 0.07	1.06 ± 0.07	1.42 ± 0.06	0.62 ± 0.03
τslow at 0 mV (ms)	nNav1.5	5.51 ± 0.24	5.14 ± 0.63	6.41 ± 0.32	7.28 ± 0.45	8.18 ± 0.51	10.31 ± 1.16	5.80 ± 0.30
	aNav1.5	5.10 ± 0.24	5.07 ± 0.45	5.60 ± 0.36	6.38 ± 0.44	8.53 ± 0.79	12.68 ± 0.97	5.92 ± 0.56
τrecovery (ms)	nNav1.5	22.9 ± 1.2	—	—	16.4 ± 1.1	—	—	24.1 ± 1.7
	aNav1.5	21.7 ± 1.1	—	—	16.7 ± 0.8	—	—	24.4 ± 1.2

For each parameter (given rows), data are shown for nNav1.5 (above) and aNav1.5 (below) for values of pH_o in the range 5.25 to 8.25. Control and recovery values (at the normal pH_o 7.25) are matched with pH_o 6.25. Data are presented as mean ± standard error of the mean (n ≥ 10). Unpaired t-tests were used to assess whether effects of H⁺ were differential for nNav1.5 versus aNav1.5.

Significance: ^*p < 0.05, ^**p < 0.01.

G_max, maximal Na⁺conductance; V_thres, threshold voltage for activation; V_peak, voltage at which Na⁺ current is maximal; V_1/2, half-maximal voltage; k, slope factor; V₀, coefficient describing the voltage dependence of time to peak; T_peak, time to peak; τ_fast, fast inactivation decay constant; τ_slow, slow inactivation decay constant; τ_recovery, recovery from inactivation time constant.

Table 2.

Summary of Electrophysiological Effects on H⁺ on Neonatal and Adult Nav1.5

Parameter	Splice variant	n	pKa	Maximum asymptote	Minimum asymptote	Hill coefficient (nH)	R
Relative Gmax (%)	nNav1.5	≥10	5.90 ± 0.14	101.69 ± 2.85	2.73 ± 13.25	0.96 ± 0.20	0.99
	aNav1.5	≥10	5.91 ± 0.14	102.07 ± 3.01	1.01 ± 13.60	0.95 ± 0.20	0.99
Δ activation V1/2 (mV)	nNav1.5	≥10	5.46 ± 0.11	41.01 ± 4.75	−0.95 ± 0.59	−1.04 ± 0.22	0.99
	aNav1.5	≥10	5.43 ± 0.05	54.69 ± 3.08*	−1.07 ± 0.36	−1.01 ± 0.14	0.99
Δ Tpeak at 0 mV (ms)	nNav1.5	≥10	5.85 ± 0.10	0.72 ± 0.07	−0.00 ± 0.04	−1.05 ± 0.04	0.97
	aNav1.5	≥10	5.83 ± 0.07	0.58 ± 0.03	−0.00 ± 0.03	−1.02 ± 0.06	0.98
Δ τfast at 0 mV (ms)	nNav1.5	≥10	5.55 ± 0.10	1.38 ± 0.15	−0.01 ± 0.01	−0.99 ± 0.10	1.00
	aNav1.5	≥10	5.48 ± 0.03	1.48 ± 0.16	−0.00 ± 0.02	−0.98 ± 0.09	0.98

For each parameter (given columns), data are shown for nNav1.5 (above) and aNav1.5 (below). pK_a values represent pH units. The maximum asymptote (maximum V_1/2 shift) for nNav1.5 was restricted at 54.7 mV (i.e., the maximum asymptote for aNav1.5). The maximum asymptote for both fits was restricted at the maximal effect occurring at pH_o 5.25. For all parameters studied, the minimum asymptotes were not statistically different from zero. Unpaired Student's t-tests were used to compare the effects on nNav1.5 and aNav1.5.

Significance ^*p < 0.05.

pK_a, pH giving half-maximum effect; R, the correlation coefficient of the mean-least-squares fit.

G_max and voltage dependence of activation

Acidifying pH_o from 7.25 (control) to 5.25 resulted in rapid inhibition of peak I_Na (Fig. 1A, B). These effects and time courses were similar for both Nav1.5 variants and recovery was complete within <20 s. The effects of H⁺ on I_Na and G_max were determined at various values of pH_o (8.25, 6.75, 6.25, 5.75, and 5.25) by changing pH_o in random order (Table 1). The alkaline shift (to pH_o 8.25) had no effect on peak I_Na, G_max or voltage- and time-dependent properties of the Nav1.5 variants (Fig. 1C, D and Table 1). As a control, we should note that the data confirmed the significantly depolarized activation voltage of nNav1.5 versus aNav1.5, reported earlier (Fig. 1D).²¹

FIG. 1.

Effects of H⁺: Time course of acid block and lack of effect of alkalinization. (A) Typical whole-cell nNav1.5 and aNav1.5 currents in pH_o 7.25 (control/wash), pH_o 6.25 and pH_o 5.25. Currents were elicited by 12 ms depolarizing pulses to −20 mV at 0.1 Hz from a holding potential of −100 mV. Traces shown were recorded at stages (a–e) indicated in (B). Acid block was fully reversible upon washout. Dashed horizontal lines indicate zero current. (B) Time course of onset and offset of acid block of nNav1.5 current (protocol as in A): a, pH_o 7.25 (control); b, pH_o 6.25; c, pH_o 7.25 (washout for pH_o 6.25); d, pH_o 5.25; e, pH_o 7.25 (washout for pH_o 5.25). Similar time course for acid block was also obtained for aNav1.5 (not shown). (C) Alkalinization (pH_o 8.25) had no effect on peak current amplitude in both Nav1.5 variants. Traces shown are typical whole-cell nNav1.5 currents, before and after exposure to pH_o 8.25. Currents were elicited by 60 ms pulses to between −80 and +45 mV from a holding potential of −100 mV; interpulse duration was 2 s. (D) Normalized current–voltage (I–V) relationships for pH_o 7.25 (control and washout) and pH_o 8.25, showing that alkalinization did not alter voltage dependence of activation of nNav1.5 or aNav1.5. All data are mean ± SEM (n = 5–12). Some error bars are smaller than symbols. SEM, standard error of the mean.

The normalized I–V relationships demonstrated that inhibition of I_Na by H⁺ involved two different effects: G_max was reduced and the activation voltage range was depolarized (Fig. 2A, B). The inhibitory effects of acidifying pH_o on G_max of nNav1.5 and aNav1.5 were statistically similar (Fig. 2A, B and Table 1). Thus, changing pH_o from 7.25 to 6.25 caused ∼30% suppression of G_max in both cases. The G_max − pH_o dose–response relationships also gave similar pK_a values (Table 2). For both channel variants, the minimum asymptotes of the fits were not statistically different from zero (Table 2), that is, there was no pH_o-independent fraction of I_Na. A similar result was obtained earlier for aNav1.5.³⁸

FIG. 2.

Effects of H⁺: Acid-induced suppression of peak conductance and depolarization of voltage dependence of activation. (A, B) Normalized peak I–V relationships at each pH_o studied (for the range 7.25 to 5.25) for nNav1.5 (A) and aNav1.5 (B). Current amplitudes were normalized to the maximal peak I_Na recorded at control pH_o (7.25). Insets, corresponding conductance transforms. All pre- and post-treatment control data (pH_o 7.25) are included to show full recovery from acidic effects and the absence of background time-dependent shifts in voltage dependence of Nav1.5 activation. (C, D) Normalized G–V relationships for each pH_o tested, fitted with Boltzmann functions for nNav1.5 (C) and aNav1.5 (D). All data are presented as mean ± SEM (n ≥ 10 cells).

To isolate effects of H⁺ on voltage dependence of activation, peak conductance was normalized to G_max for each value of pH_o, and data were fit to the Boltzmann function (Eq. 1) on a cell-by-cell basis (Fig. 2C, D). For decreasing pH_o, progressive depolarization of activation-V_1/2 and slope factor k increases were observed. For both channel variants, these effects were statistically significant for changes in pH_o from 7.25 to ≤6.25 (p < 0.05 vs. control; Fig. 2C, D and Table 1). Importantly, these changes were significantly smaller for nNav1.5 versus aNav1.5 at pH_o ≤ 5.75 (Table 1). Consistent with these differential effects, logistic fits to the activation-V_1/2 data yielded a significant (∼14 mV) larger maximum asymptote for aNav1.5 versus nNav1.5 (Table 2). In contrast, pK_a values were similar (Table 2).

Threshold of activation (V_thres) and voltage for peak I_Na (V_peak) were also depolarized in acidified solutions (Fig. 2A, B and Table 1). Shifts in V_thres were statistically significant for change of pH_o to ≤6.25 (p < 0.05 vs. control); however, there was no difference between the two Nav1.5 variants (Table 1). Shifts in V_peak, which were also statistically significant for pH_o ≤ 6.25 (p < 0.05 vs. control), were larger for aNav1.5 versus nNav1.5 at pH_o 5.75 and 5.25 (Table 1). Finally, block of I_Na by acidification (studied at pH_o 6.25) was voltage dependent for both Nav1.5 variants, the effect being significantly greater at negative potentials, peaking at −45 mV for both. It is possible that the accompanying positive shifts in activation gating (in particular V_thres) contributed to this effect. Nevertheless, in the voltage range −45 to −55 mV, this effect of voltage was smaller by 17–57% for nNav1.5 versus aNav1.5 (p < 0.05 for all).

Kinetics of activation and inactivation

Activation and inactivation kinetics were slowed in acid pH_o (Table 1). For both Nav1.5 variants, the slowing of time to peak (T_peak) was significant for pH_o ≤ 6.25 but similar for both Nav1.5 variants (Table 1). Logistic fits to the acid-induced shifts at 0 mV produced comparable pK_a values (Table 2). The voltage dependence of T_peak was not altered (p > 0.05 vs. control; Table 1). Double exponential fits (Eq. 3) to I_Na decays suggested that currents decayed more slowly at pH_o ≤ 6.25. Thus, fast (τ_fast) and slow (τ_slow) time constants of inactivation were increased, but this was similar for both variants (Table 1).

Availability and recovery from inactivation

Acidifying pH_o depolarized the inactivation-V_1/2 significantly but similarly for both Nav1.5 variants (p < 0.05 vs. control for both), whereas the slope factor was not affected (Fig. 3A, C, E and Table 1). Overall, acid-induced shifts in inactivation-V_1/2 were smaller than for activation-V_1/2 (Table 1). Acidosis significantly quickened recovery from inactivation (τ_recovery) (p < 0.05 vs. control); this was similar for both splice variants (Fig. 3B, D, F and Table 1).

FIG. 3.

Effects of H⁺: Acid-induced depolarization of steady-state inactivation and acceleration of recovery from inactivation. (A) Typical whole-cell nNav1.5 currents in pH_o 7.25 (control) and pH_o 6.25. (B) Superimposed whole-cell Na⁺ currents from nNav1.5 recovery from inactivation, in pH_o 7.25 (i) and pH_o 6.25 (ii). Arrows indicate acid-induced acceleration of current recovery. (C, E) Availability-voltage relationships in pH_o 7.25 (control/wash), pH_o 6.25, and pH_o 5.25 fitted with Boltzmann functions for nNav1.5 (C) and aNav1.5 (E). (D, F) Recovered current (I_t/I_c) in pH_o 7.25 (control/wash) and pH_o 6.25, plotted as a function of stimulation interval and fitted with single exponential functions for nNav1.5 (D) and aNav1.5 (F). Significance (paired t-tests vs. control): *p < 0.05, **p < 0.01. Data are mean ± SEM. (n = 10–19).

Discussion

In this study, the electrophysiological effects of H⁺ were compared for “adult” versus “neonatal” splice variants of Nav1.5. The main findings were as follows: (1) H⁺ inhibited peak I_Na through reducing G_max and inducing a positive shift in voltage dependence of activation. (2) Proton block of G_max was similar for the two Nav1.5 variants, but the acid-induced depolarization of voltage dependence of activation was significantly smaller for nNav1.5 compared with aNav1.5. These results are discussed in relation to possible mode(s) of action of H⁺ and the difference in the charged amino acids of nNav1.5 versus aNav1.5.

Acid block of I_Na has been studied in native cardiomyocytes, neurons, skeletal muscle as well as recombinantly expressed VGSCs.^{24,25,29–31,38–43} Consistent with the results presented in this study, two distinct effects of extracellular acidosis on VGSCs have been routinely observed: reduction of G_max and depolarization of voltage dependence of channel gating. In addition, effects on slow and use-dependent inactivation, not determined in this study, have been reported for Nav1.2/1.4/1.5.^{29–31,41,42}

The precise mechanism(s) of acid block of Na⁺ conductance is not understood. For Nav1.5, this is thought to involve four outer-ring amino acids (i.e., the “EEDD” motif) and the “TTX-sensitivity determining” Cys³⁷³ located just above the selectivity-filter in DI.^30,38,44 In addition, His⁸⁸⁰, located at the top of the P1 helix of DII, has been found to contribute to modulation of slow inactivation kinetics and use-dependent inactivation by acidosis.³⁰ In contrast, selectivity-filter carboxyl groups (“DEKA” motif) were found to be protected from protonation possibly because of their proximity to the positive lysine residue (K) present.^38,44

In previous studies, no pH_o-independent fraction of Na⁺ conductance was found for Nav1.5 (unlike for Nav1.4).^30,38 This was also found in this study and could be due, in part, to the protonation of Cys³⁷³ in DI.^30,38 Also, proton inhibition of peak Na⁺ conductance has been shown to be well represented by a single-site binding model.^38,40 This was also found with block of G_max being fit best by a Hill equation for a single site. This gave pK_a values of ∼5.9 for both nNav1.5 and aNav1.5, which compare well with previously reported values for aNav1.5.^29,30,38 Similar values of pK_a were found for Nav1.4 and shown to be independent of β1 subunit co-expression.^40,44,45

The similarity in the values of pK_a for G_max of nNav.5 and aNav1.5 is consistent with the spliced DI:S3/S4 region being located peripheral to the outer vestibule of the channel pore, as suggested by X-ray crystal structures of the bacterial sodium channel and the mammalian voltage-gated K⁺ channel.^46,47 Furthermore, mutagenesis of the outer ring negative carboxylates in various VGSCα isoforms to neutral or positive residues had no detectable effect on voltage dependence of activation, again suggesting that channel voltage sensors are distant from the outer vestibule residues.^44,45,48,49 Taken together, therefore, the spliced DI:S3/S4 region (including the charge-reversing Lys-211-Asp) must be sufficiently away from the channel outer vestibule that the amino acid changes between nNav1.5 and aNav1.5 do not appear structurally or electrostatically to modify the acidic inhibition of Na⁺ permeation.

As regards channel gating, acid pH_o depolarized steady-state activation and inactivation, slowed activation and inactivation kinetics, and accelerated recovery from inactivation in both Nav1.5 variants. These results agree with previous studies.^24,38,45,50 On the whole, the observed effects of protons on VGSC gating may be due to screening or titrating the negatively charged sialic acids and carboxylates on the channel surface, resulting in (1) reduction of negative surface potential and (2) positive shift in activation voltage.¹ However, other mechanism(s), such as direct channel block, may also be involved.^1,24,40,44

Importantly, the pH_o dependence of activation differed significantly between the two variants. Thus, acidifying pH_o to ≤5.75 shifted activation V_1/2, k, and V_peak of aNav1.5 significantly more than nNav.15 (Table 1). These results are consistent with our hypothesis that substitution of the negatively charged Asp²¹¹ to the positive Lys²¹¹ in the DI:S3–S4 region (close to the voltage sensor) should reduce the apparent effect of protonation on voltage dependence of activation of nNav1.5. In agreement with this, earlier studies also have reported that acidic residues in S3–S4 linkers are important determinants of cationic effects on channel gating.^51,52

Conclusion

We hypothesized that H⁺ effects on channel gating would be smaller for nNav1.5 versus aNav1.5, since the negative surface potential near D1:S4 may already be partly reduced due to the Asp²¹¹ to Lys charge reversal in nNav1.5. Taken together, the data clearly indicate that nNav1.5 is more acid resistant than aNav1.5. This has important implications regarding the involvement of nNav1.5 in cancer. First, many tumors grow under hypoxia, resulting in acidic conditions.⁵³ Second, in the breast cancer cell line MDA-MB-231, nNav1.5 activity leads to an intracellular alkalinization and pericellular acidification favoring increased protease activity, enhancing invasiveness.⁵⁴ Thus, expression of nNav1.5 (vs. aNav1.5) would mean better maintained VGSC activity within the acidic environment, enhancing its contribution to metastatic cell behaviors.¹² Finally, low pH_o also increases the late (“persistent”) Na⁺ current critical to metastatic cell behaviors.⁵⁵ Ranolazine, a specific inhibitor of the persistent Na⁺ currents, is currently clinically approved for treatment of angina pectoris.⁴¹ Importantly, ranolazine has been shown to work in a pH-dependent manner on Nav1.5, with increasing effectiveness at acidic pH.⁴¹ Thus, such a drug may have potential as an anti-metastatic agent.⁵⁵

Authors' Contributions to and Responsibilities for the Article

M.B.A.D. and R.O. conceived the research. S.P.F. and R.O. performed the experiments and did the analyses. All authors contributed to writing the article. All coauthors have reviewed and approved of the article before submission.

This article has been submitted solely to Bioelectricity and is not published, in press, or submitted elsewhere.

Author Disclosure Statement

M.B.A.D. is involved in a spinout company (Celex Oncology Ltd) focused on ion channels and cancer. S.P.F. and R.O. declare no competing financial interests.

Funding Information

We thank Pro Cancer Research Fund (PCRF) for continuous support (M.B.A.D.; S.P.F.). R.O. was funded by a PhD studentship from the British Heart Foundation (BHF).