Reduced sodium channel density, altered voltage dependence of inactivation, and increased susceptibility to seizures in mice lacking sodium channel beta 2-subunits

Abstract

Sodium channel beta-subunits modulate channel gating, assembly, and cell surface expression in heterologous cell systems. We generated beta2(-/-) mice to investigate the role of beta2 in control of sodium channel density, localization, and function in neurons in vivo. Measurements of [(3)H]saxitoxin (STX) binding showed a significant reduction in the level of plasma membrane sodium channels in beta2(-/-) neurons. The loss of beta2 resulted in negative shifts in the voltage dependence of inactivation as well as significant decreases in sodium current density in acutely dissociated hippocampal neurons. The integral of the compound action potential in optic nerve was significantly reduced, and the threshold for action potential generation was increased, indicating a reduction in the level of functional plasma membrane sodium channels. In contrast, the conduction velocity, the number and size of axons in the optic nerve, and the specific localization of Na(v)1.6 channels in the nodes of Ranvier were unchanged. beta2(-/-) mice displayed increased susceptibility to seizures, as indicated by reduced latency and threshold for pilocarpine-induced seizures, but seemed normal in other neurological tests. Our observations show that beta2-subunits play an important role in the regulation of sodium channel density and function in neurons in vivo and are required for normal action potential generation and control of excitability.

Fig 1.

[³H]STX-binding analysis of membrane preparations and primary cultured neurons from β2^+/+ and β2^−/− mice. (A) Specific [³H]STX binding to brain membranes from β2^+/+ and β2^−/− mice. Error bars show SEM. (B) Specific [³H]STX binding to primary cultures from β2^+/+ and β2^−/− mouse brains. Error bars show SEM; *, P = 0.0245. (C) Western blot analysis. Lane A, rat brain membranes; lane B, neurons from β2^+/+ mouse B326; lane C, neurons from β2^+/+ mouse B392; lane D, neurons from β2^+/+ mouse B368; lane E, neurons from β2^−/− mouse A10.

Fig 2.

Sodium currents in acutely dissociated hippocampal neurons from β2^−/− mice. (A) Mean current-voltage relationships for β2^+/+ (•) and β2^−/− (○) mice from a holding potential of −80 mV normalized to the peak current and averaged (β2^+/+, n = 12; β2^−/−, n = 10). (Inset) Example current traces at −10 mV from β2^+/+ (larger) and β2^−/− (smaller) mice. (B) Mean peak sodium current was measured at the minimum of the current–voltage relationship (−25 to −30 mV) and normalized to cell capacitance from neurons dissociated from β2^+/+ (n = 12 cells from six mice), β2^−/− H5 (n = 8 cells from three mice), and β2^−/− C10 (n = 11 cells from six mice) lines. Cell capacitance did not differ between groups. (C) Mean conductance-voltage relationships. Conductance-voltage data from individual experiments were fit with a Boltzmann relationship (see Experimental Procedures). For β2^−/− C10 cells, V_a = −41.0 ± 1.6 mV, k = 6.95 ± 0.47 mV (n = 10); for β2^+/+, V_a = −42.3 ± 1.27 mV, k = 6.36 ± 0.39 mV (n = 8). (D) Steady-state inactivation of sodium currents in β2^+/+ and β2^−/− neurons. The average half inactivation voltage for β2^−/− C10 neurons was −67.5 ± 2.78 mV, k = 7.22 ± 0.90 mV (n = 10), and for β2^+/+ neurons, it was −56.9 ± 2.46, k = 8.25 ± 0.72 mV (n = 10; P < 0.01).

Fig 3.

Reduced sodium current in optic nerves of β2^−/− mice. (A) Compound action potentials for β2^+/+, β2^−/−, and normalized β2^−/−. (B) Latency from stimulus to action potential recording for each peak in the compound action potential. (C–F) The areas of optic nerve compound action potentials are plotted as a function of stimulus intensity. (C) β2^+/+ in 140 mM Na⁺: •, control; ○, 10 nM TTX; ••••, 10 nM TTX normalized (n = 3). (D) β2^+/+: •, control; ○, 40 mM Na⁺; ••••, 40 mM Na⁺ normalized (n = 16). (Bars = 1 mV, 0.5 ms.) (E) •, β2^+/+, 140 mM Na⁺ (n = 16); ○, β2^−/− C10, 140 mM Na⁺; ••••, β2^−/− C10 normalized (n = 16). (F) •, β2^+/+, 40 mM Na⁺ (n = 16); ○, β2^−/− C10, 40 mM Na⁺; ••••, β2^−/− C10 normalized (n = 16). CAP, compound action potential area in mV/ms.

Fig 4.

Sodium channel clustering and node of Ranvier formation in β2^−/− mice. Blue, MAG; green, sodium channels; red, Caspr. (A) Nodes of Ranvier in adult sciatic axons from β2^−/− and β2^+/+ mice. (B) Sodium channel clustering and formation of new nodes of Ranvier during remyelination, 14 days after injection. sing, single Schwann cell processes and associated sodium channel clusters; bin, binary sets of clusters; focal, new nodes with focal clusters of sodium channels. (C) Nodes of Ranvier in adult optic nerves of β2^+/+ and β2^−/− mutant mice. [Bars = 5 μm (A and B); 10 μm (C).]

Fig 5.

Localization of Na_v1.2 and Na_v1.6 in optic nerves from β2^−/− mice. (A–C) Optic nerve fibers from β2^−/− mice double-labeled with anti-Nav1.6 (A) and anti-pan sodium channel (B) antibodies illustrating colocalization of these antibodies at the nodes of Ranvier of optic nerves (C). Regions of overlap are shown in yellow and yellow/orange. (D–F) Optic nerve fibers from β2^+/+ mice double-labeled with anti-Na_v1.6 (D) and anti-pan sodium channel (E) illustrating colocalization (F) of these antibodies in the optic nerve. (G–I) Optic nerve fibers from β2^+/+ mice double-labeled with anti-Na_v1.2 (G) and anti-pan sodium channel (H) antibodies. Regions of overlap would appear yellow in the merged image (I). (J–L) Optic nerves from β2^−/− mice double-labeled with anti-Na_v1.2 (G) and anti-pan sodium channel (H) antibodies. Regions of overlap would appear yellow in the merged image (L).

Fig 6.

Pilocarpine-induced stage 3 seizures in WT and β2^−/− mice. (A) The frequency of prolonged seizures induced by 340 mg/kg pilocarpine was different in F₂ hybrid β2^+/+ and β2^−/− mice. The β2^−/− mice exhibited an increased frequency of stage 3 (forelimb clonus) or higher seizure activity compared with β2^+/+ mice (*, P < 0.05, χ² analyses). (B) Latency to onset of pilocarpine-induced stage 3 seizures in F₂ hybrid β2^+/+ and β2^−/− mice. The latency to stage 3 seizures induced by 340 mg/kg pilocarpine was different in β2^+/+ and β2^−/− mice. The β2^−/− mice had a decreased latency to stage 3 seizures compared with β2^+/+ mice (*, P < 0.05, Student's t test). The number of mice tested is shown above each bar. (C) Seizure frequency after injection of 340 mg/kg of pilocarpine as in A for N₁₀ generation mice in the C57BL/6J genetic background. (Bars represent SEM.) **, P < 0.01.