The sodium channel Nav1.5a is the predominant isoform expressed in adult mouse dorsal root ganglia and exhibits distinct inactivation properties from the full-length Nav1.5 channel

Abstract

Nav1.5 is the principal voltage-gated sodium channel expressed in heart, and is also expressed at lower abundance in embryonic dorsal root ganglia (DRG) with little or no expression reported postnatally. We report here the expression of Nav1.5 mRNA isoforms in adult mouse and rat DRG. The major isoform of mouse DRG is Nav1.5a, which encodes a protein with an IDII/III cytoplasmic loop reduced by 53 amino acids. Western blot analysis of adult mouse DRG membrane proteins confirmed the expression of Nav1.5 protein. The Na+ current produced by the Nav1.5a isoform has a voltage-dependent inactivation significantly shifted to more negative potentials (by approximately 5 mV) compared to the full-length Nav1.5 when expressed in the DRG neuroblastoma cell line ND7/23. These results imply that the alternatively spliced exon 18 of Nav1.5 plays a role in channel inactivation and that Nav1.5a is likely to make a significant contribution to adult DRG neuronal function.

Fig. 1

(A) Expression of Na_v1.5 mRNA isoforms in adult and neonatal mouse dorsal root ganglia (DRG). A faint Na_v1.5/Na_v1.5c product of approximately 520 bp and a more abundant Na_v1.5a (Δ exon 18) product of approximately 360 bp were detected in adult (lane 3) and neonatal DRG (lane 5) RNA that had been reverse transcribed (RT⁺), but not from either adult or neonatal DRG RNA that has not been reverse transcribed (RT^-; lanes 2 and 4, respectively. Lane 1 was a 1 kb DNA ladder (Invitrogen). The neonatal sample was from postnatal day 2 (P2), and RT-PCR was for 35 cycles. (B) Expression of Na_v1.5 mRNA isoforms in adult rat tissues. Na_v1.5/Na_v1.5c and Na_v1.5a products of approximately 370 and 210 bp were detected in adult rat DRG (lane 3), trigeminal ganglia (lane 4) and heart (lane 5). Lane 2 was a water control, and lane 1 was a 1 kb DNA ladder. RT-PCR was for 35 cycles.

Fig. 2

Expression of Na_v1.5 and Na_v1.8 mRNAs decrease in adult mouse DRG after axotomy. In quantitative RT-PCR assays the expression of Na_v1.5a mRNA decreased to 0.519±0.027 of control (n=5; P<0.0005), Na_v1.5/Na_v1.5c mRNA decreased to 0.536±0.019 of control (n=6; P<0.0001) and Na_v1.8/Na_v1.8c mRNA decreased to 0.536±0.065 of control (n=6; P<0.0005) in pooled ipsilateral (axotomized) lumbar L4 and L5 DRG compared to contralateral (unaxotomized) controls, 7 days after axotomy. Data are shown as mean±S.E., in which expression after axotomy (filled boxes) was compared to contralateral controls of 1.00 relative units (unfilled boxes).

Fig. 3

Detection of Na_v1.5 protein in DRG and heart membrane proteins isolated from adult mice. A Western blot of detergent-solubilized membrane proteins from heart (50 μg, lane 1) and DRG (400 μg, lane 3) was probed with an anti-Na_v1.5 antibody, resulting in the detection in both tissues of a single major band with an apparent molecular weight (M_r)of >250 kDa. Lane 2 is blank. Positions of molecular weight markers are indicated in kDa.

Fig. 4

Voltage-gated inward currents through Na_v1.5 and Na_v1.5a channels. (A) Example of inward currents through Na_v1.5 channels activated by depolarizing pulses to the command potentials (CP) indicated. Holding potential was −92 mV. Solid line superimposed on current trace at −47 mV shows an example of a fit to a decaying exponential (Eq. (4)). Inset shows voltage protocol. (Identical findings were observed using the Na_v1.5a construct.) (B) Peak inward current–voltage relation for data shown in A. Open circles—uncorrected data; filled triangles—data corrected for voltage-drop across the series resistance (Eq. (1)). Solid lines represent a fit to a modified Boltzmann relation (Eq. (2)). In this representative example, V_0.5,act for uncorrected data shown was −46.5 mVand for corrected data was −41.6 mV. (C) Mean (±S.E.M.) V_0.5,act for Na_v1.5 and Na_v1.5a channel currents. (D) Mean (±S.E.M.) for slope factors (s). *, P<0.05.

Fig. 5

Inactivation of Na_v1.5 and Na_v1.5a channel currents. (A) Voltage-dependence of time constant (τ; Eq. (4)) of inactivation for Na_v1.5 (open circles) and Na_v1.5a (filled triangles). ***, P<0.001; *, P<0.05. (B) Recovery from inactivation of Na_v1.5 (open circles) and Na_v1.5a (filled triangles) channel currents. Solid lines represent fits to double exponential relation (Eq. (5)). Fitted parameters were as follows: Na_v1.5, A=0.96±0.05, τ₁=5.04±0.24 ms, τ₂=158.9±28.8 ms (n=28); Na_v1.5a, A=0.97±0.04, τ₁=4.52±0.22 ms, τ₂=246.9±60.3 ms (n=28, n.s.). (C) Representative current traces obtained at a command potential of −22 mV following preconditioning potentials indicated using voltage protocol shown. (D) Left-hand panel shows mean (±S.E.M.) V_0.5,inact for Na_v1.5 and Na_v1.5a channel currents. **, P<0.01. Right-hand panel shows mean (±S.E.M.) for slope factors (s).

Fig. 6

Voltage-dependence of activation and inactivation for Na_v1.5 and Na_v1.5a currents. The mean fitted parameters used are from Figs. 4C, D and 5D. Na_v1.5: V_0.5,act=−41.1 mV, s_act=5.8 mV; V_0.5,inact=−83.3 mV, s_inact=−5.5 mV. Na_v1.5a: V_0.5,act=−40.2 mV, s_act=6.6 mV; V_0.5,inact=−88.1 mV, s_inact=−5.5 mV. Inset shows region of overlap between activation and inactivation that would produce a window current. Arrows indicate voltage of peak window current for each isoform.