Aminopyridines potentiate synaptic and neuromuscular transmission by targeting the voltage-activated calcium channel beta subunit

Abstract

Aminopyridines such as 4-aminopyridine (4-AP) are widely used as voltage-activated K(+) (Kv) channel blockers and can improve neuromuscular function in patients with spinal cord injury, myasthenia gravis, or multiple sclerosis. Here, we present novel evidence that 4-AP and several of its analogs directly stimulate high voltage-activated Ca(2+) channels (HVACCs) in acutely dissociated neurons. 4-AP, 4-(aminomethyl)pyridine, 4-(methylamino)pyridine, and 4-di(methylamino)pyridine profoundly increased HVACC, but not T-type, currents in dissociated neurons from the rat dorsal root ganglion, superior cervical ganglion, and hippocampus. The widely used Kv channel blockers, including tetraethylammonium, alpha-dendrotoxin, phrixotoxin-2, and BDS-I, did not mimic or alter the effect of 4-AP on HVACCs. In HEK293 cells expressing various combinations of N-type (Cav2.2) channel subunits, 4-AP potentiated Ca(2+) currents primarily through the intracellular beta(3) subunit. In contrast, 4-AP had no effect on Cav3.2 channels expressed in HEK293 cells. Furthermore, blocking Kv channels did not mimic or change the potentiating effects of 4-AP on neurotransmitter release from sensory and motor nerve terminals. Thus, our findings challenge the conventional view that 4-AP facilitates synaptic and neuromuscular transmission by blocking Kv channels. Aminopyridines can directly target presynaptic HVACCs to potentiate neurotransmitter release independent of Kv channels.

FIGURE 1.

4-AP stimulates HVACC currents in acutely dissociated neurons. A, representative traces and I-V curves of I_Ba before and during application of 5 mm 4-AP in DRG neurons (n = 16). Neurons were voltage-clamped at −90 mV and depolarized from −70 to 50 mV for 150 ms with 10 mV increments. *, p < 0.05 compared with corresponding values before 4-AP application. B, steady-state activation curves of I_Ba before and during 4-AP application in 16 DRG neurons. Before 4-AP application, the V_0.5 and slope factor were −12.7 ± 0.2 and 5.0 ± 0.2 mV, respectively. During 4-AP application, the V_0.5 and slope factor were −18.0 ± 0.1 and 4.6 ± 0.1 mV, respectively. C, representative current traces and summary data showing steady-state inactivation of I_Ba before and during 4-AP application in nine DRG neurons. The V_0.5 values were −46.5 ± 1.2 and −38.0 ± 1.5 mV (p < 0.05) before and during 4-AP application, respectively. The slope factors were −13.3 ± 1.2 and −18.9 ± 2.2 mV (p < 0.05) before and during 4-AP application, respectively. Cells were depolarized at a series of prepulse potentials (from −90 to 10 mV, 500 ms) and then depolarized to 0 mV. The normalized conductance (G/G_max) was fit to the Boltzmann function. D, summary data showing the effects of 4-AP on I_Ba in SCG and hippocampal neurons.

FIGURE 2.

Comparison of the concentration-response effects of 4-AP on Kv channel currents and I_Ba in DRG neurons. A, original traces and summary data show the concentration-dependent effects of 4-AP on Kv channel currents in DRG neurons (n = 6). B, average data show the concentration-dependent effects of 4-AP on I_Ba in DRG neurons (n = 10). *, p < 0.05 compared with the corresponding control.

FIGURE 3.

Effects of 4-AP on the subtypes of HVACC currents. A, average data show that cadmium (Cd²⁺) blocked I_Ba and the effect of 5 mm 4-AP on I_Ba in DRG neurons. B, summary data show that 4-AP increased both the total and N-type Ca²⁺ I_Ba in DRG neurons (n = 6). C, summary data show the effect of 4-AP on the total and L-type I_Ba in DRG neurons (n = 6). N- and L-types represent pharmacologically isolated N- and L-type Ca²⁺ channel currents. D, summary data show a lack of effect of 4-AP on T-type I_Ba in DRG neurons (n = 7). *, p < 0.05 compared with the corresponding control; #, p < 0.05 compared with the isolated N- or L-type I_Ba amplitude. pF, picofarad.

FIGURE 4.

4-AP stimulates HVACCs in DRG neurons independent of Kv channels. A, original traces and summary data show that 140 mm TEA had no effect on I_Ba or the effect of 4-AP on I_Ba. B, representative current traces and summary data show that the effect of 4-AP on I_Ba was not altered in the presence of 100 nm α-dendrotoxin (α-DTX). C, summary data show that 1 μm phrixotoxin (PhrixoTx) or 1 μm BDS-I did not mimic or alter the effect of 4-AP on I_Ba. D, original current traces and summary data show that 2 μm KN-93 did not change 4-AP-induced augmentation of I_Ba. *, p < 0.05 compared with the corresponding value during the control or washout (c/w).

FIGURE 5.

Comparison of the effects of aminopyridines and 4-AP analogs on HVACCs in DRG neurons. A, chemical structure of aminopyridines and 4-AP analogs. B, comparison of the concentration-response effects of four aminopyridines on the peak amplitude of I_Ba. C, comparison of the concentration-response effects of four 4-AP analogs on I_Ba. 2-AP, 2-aminopyridine; 3-AP, 3-aminopyridine; 4-AMP, 4-(aminomethyl)pyridine; 4-MAP, 4-(methylamino)pyridine; 4-A-2-MP, 4-amino-2-methylpyridine; 4-DAMP, 4-di(methylamino)pyridine.

FIGURE 6.

4-AP stimulates N-type (Cav2. 2) HVACCs through the β₃ subunit expressed in HEK293 cells. A, raw current traces and time course showing that 5 mm 4-AP increased N-type I_Ba. B, current traces and I-V curves showing that 4-AP increased N-type I_Ba. Cells were voltage-clamped at −90 mV and depolarized from −70 to 50 mV for 150 ms with 10-mV increments. *, p < 0.05 compared with corresponding values before 4-AP application. C, comparison of the effects of 4-AP on N-type I_Ba reconstituted with different combinations of N-type Ca²⁺ channel subunits. *, p < 0.05 compared with the effect of 4-AP on I_Ba without the β₃ subunit. D, steady-state activation curves of N-type I_Ba (α₁B + β₃ + α₂δ) before and during 4-AP application in 12 HEK293 cells. Before 4-AP application, the V_0.5 and slope factor were −2.3 ± 0.1 and 3.6 ± 0.1 mV, respectively. During 4-AP application, the V_0.5 and slope factor were −7.8 ± 0.3 and 4.0 ± 0.3 mV, respectively. Neurons were voltage-clamped at −90 mV and depolarized from −70 to 20 mV for 150 ms with 10-mV increments. E, original current traces and summary data showing that 4-AP shifted the steady-state inactivation curve of N-type I_Ba (α₁B + β₃ + α₂δ) toward the left (n = 8). The V_0.5 and slope factor before 4-AP application were −62.3 ± 0.4 and −6.3 ± 0.3 mV, respectively. After 4-AP, the V_0.5 and slope factor were −54.0 ± 0.3 and −5.8 ± 0.3 mV, respectively. F, current traces and I-V curves showing the lack of effect of 4-AP on T-type (Cav3.2) Ca²⁺ channels expressed in HEK293 cells. w, washout; c, control; pF, picofarad.

FIGURE 7.

4-AP increases glutamate release from primary afferent terminals in the spinal cord independent of Kv channels. A, original traces and summary data show the concentration-dependent effect of 4-AP on evoked monosynaptic glutamatergic EPSCs of spinal dorsal horn neurons. B, representative recordings and summary data show that 100 nm α-dendrotoxin (DTX) and 3 μm CP339818 (CP) had no significant effect on 4-AP-induced increases in the amplitude of EPSCs of dorsal horn neurons. *, p < 0.05 compared with the control (Cont); #, p < 0.05 compared with the effect of α-dendrotoxin and CP339818 alone.

FIGURE 8.

4-AP potentiates acetylcholine release from motor nerve terminals in the phrenic nerve-diaphragm independent of Kv channels. A, original traces and summary data show the effect of 0.5 mm 4-AP on evoked cholinergic EPPs. B, representative recordings and summary data show that 100 nm α-dendrotoxin (DTX) and 3 μm CP339818 (CP) had no significant effect on 4-AP-induced potentiation of EPPs. *, p < 0.05 compared with the control (Cont); #, p < 0.05 compared with the effect of α-dendrotoxin and CP339818 alone.