Late Na+ current produced by human cardiac Na+ channel isoform Nav1.5 is modulated by its beta1 subunit

Abstract

Experimental data accumulated over the past decade show the emerging importance of the late sodium current (I(NaL)) for the function of both normal and, especially, failing myocardium, in which I(NaL) is reportedly increased. While recent molecular studies identified the cardiac Na(+) channel (NaCh) alpha subunit isoform (Na(v)1.5) as a major contributor to I (NaL), the molecular mechanisms underlying alterations of I(NaL) in heart failure (HF) are still unknown. Here we tested the hypothesis that I(NaL) is modulated by the NaCh auxiliary beta subunits. tsA201 cells were transfected simultaneously with human Na(v)1.5 (former hH1a) and cardiac beta(1) or beta(2) subunits, and whole-cell patch-clamp experiments were performed. We found that I(NaL) decay kinetics were significantly slower in cells expressing alpha + beta(1) (time constant tau = 0.73 +/- 0.16 s, n = 14, mean +/- SEM, P < 0.05) but remained unchanged in cells expressing alpha + beta(2) (tau = 0.52 +/- 0.09 s, n = 5), compared with cells expressing Na(v)1.5 alone (tau = 0.54 +/- 0.09 s, n = 20). Also, beta(1), but not beta(2), dramatically increased I(NaL) relative to the maximum peak current, I(NaT) (2.3 +/- 0.48%, n = 14 vs. 0.48 +/- 0.07%, n = 6, P < 0.05, respectively) and produced a rightward shift of the steady-state availability curve. We conclude that the auxiliary beta(1) subunit modulates I(NaL), produced by the human cardiac Na(+) channel Na(v)1.5 by slowing its decay and increasing I(NaL) amplitude relative to I(NaT). Because expression of Na(v)1.5 reportedly decreases but beta(1) remains unchanged in chronic HF, the relatively higher expression of beta(1) may contribute to the known I(NaL) increase in HF via the modulation mechanism found in this study.

Fig. 1

Confocal microscopy of the live tsA201 cells transfected with Na_v1.5 + β₁ (a) or Na_v1.5 + β₂ (b). Fluorescence of GFP linked to β subunit C-terminus at the cell membrane is evident for both β subunits. Optical slices were 0.5 μm (Zeiss Axiovert 100, Bio-Rad MRC 1024, excitation/emission wavelength 488/522 nm, laser power 10%)

Fig. 2

Experimental approach used to elucidate I _NaL of heterologously expressed Na_v1.5. a, b Typical examples of the I _Na currents recorded in tsA201 cells transiently transfected by α + β₁-GFP. Shown are averaged traces from 50 sweeps before and after TTX (25 μM) application to assess “zero” current. c Difference I _NaL current obtained by subtraction of “zero” current. Note different currents and time scales in a, b, and c to demonstrate peak (I _NaT) and I _NaL. V _h = −120 mV, V _m = −30 mV, 23°C

Fig. 3

Effects of the β subunits on the I _NaL produced by the heterologously expressed human cardiac NaCh isoform α subunit (Na_v1.5). a Representative examples of superimposed current traces recorded in tsA201 cells transfected with cDNA encoding human cardiac NaCh α-subunit alone or together with β₁ (α + β₁) or β₂ (α + β₂). Shown are averaged (10–20) currents with a single-exponential fit to I _NaL decay (solid lines) starting 200 ms after the onset of depolarization. The time constant (τ) values are given in the panel. The current amplitudes are relative to the peak I _NaT. The voltage-clamp procedure given in the inset. b, c Statistical analysis of the I _NaL/I _NaT ratio (b) and the decay time course (c) changes in response to coexpression of α with β subunits. The statistically significant difference (P) in panels b and c was evaluated by ANOVA followed by the Bonferroni’s post hoc test. Bars in panels b and c represent means ± SE, n number of cells. There was no significant difference between α + β₁-GFP compared with α + β₁ (1:5 cDNA ratio) or for α alone compared with α + β₂-GFP for both b and c panels

Fig. 4

Effect of β₁ subunit on the steady-state inactivation (SSI) voltage-dependency of the heterologous expressed Na_v1.5. a, b Representative raw current recordings at −30 mV in response to a different 2-s-long prepulse potential (voltage procedure is shown in c). c Data points of the relative (normalized to maximum) peak I _NaT measured at −30 mV and plotted against prepulse voltage (V _p). Solid lines represent the Bolzmann fit (Eq. 2 in “Methods”), and values are given on the plots. Statistics for values evaluated in numerous cells are given in Table 1. V _h = −140 mV, [Na]₀ = 140 mM, 24°C

Fig 5

Voltage–current relationship for heterologous expressed Na_v1.5 (α alone a, or with the β₁ subunit b). a, b Left panels raw current recordings at different membrane potentials, right panels data point plots, with the fits (solid lines) to Eq. 1 (“Methods”). The equation is given in a, and fit values for the steady-state activation are indicated on the plots in a and b. The statistical data for the steady-state activation are given in Table 1