Inhibition of neuronal degenerin/epithelial Na+ channels by the multiple sclerosis drug 4-aminopyridine

Abstract

The voltage-gated K(+) (Kv) channel blocker 4-aminopyridine (4-AP) is used to target symptoms of the neuroinflammatory disease multiple sclerosis (MS). By blocking Kv channels, 4-AP facilitates action potential conduction and neurotransmitter release in presynaptic neurons, lessening the effects of demyelination. Because they conduct inward Na(+) and Ca(2+) currents that contribute to axonal degeneration in response to inflammatory conditions, acid-sensing ion channels (ASICs) contribute to the pathology of MS. Consequently, ASICs are emerging as disease-modifying targets in MS. Surprisingly, as first demonstrated here, 4-AP inhibits neuronal degenerin/epithelial Na(+) (Deg/ENaC) channels, including ASIC and BLINaC. This effect is specific for 4-AP compared with its heterocyclic base, pyridine, and the related derivative, 4-methylpyridine; and akin to the actions of 4-AP on the structurally unrelated Kv channels, dose- and voltage-dependent. 4-AP has differential actions on distinct ASICs, strongly inhibiting ASIC1a channels expressed in central neurons but being without effect on ASIC3, which is enriched in peripheral sensory neurons. The voltage dependence of the 4-AP block and the single binding site for this inhibitor are consistent with 4-AP binding in the pore of Deg/ENaC channels as it does Kv channels, suggesting a similar mechanism of inhibition in these two classes of channels. These findings argue that effects on both Kv and Deg/ENaC channels should be considered when evaluating the actions of 4-AP. Importantly, the current results are consistent with 4-AP influencing the symptoms of MS as well as the course of the disease because of inhibitory actions on Kv and ASIC channels, respectively.

FIGURE 1.

4-AP decreases endogenous acid-sensing Deg/ENaC currents in neurons. a, structure of pyridine and its 4-amino and 4-methyl derivatives. b and c, overlay of representative traces (b) and the corresponding summary graph (c) showing the effects of 10 mm 4-AP on macroscopic acid-sensing Na⁺ currents in rat hippocampal CA1 interneurons. Traces before, after, and following wash of 4-AP are black, red, and gray, respectively. Inward current is downward with currents activated with pH5 (noted with bar). Membrane potential was clamped to −60 mV. Physiological bath and pipette solution were used in these experiments. *, significant decrease compared with before 4-AP. d, macroscopic PPK1 currents in a Drosophila class IV md peripheral sensory neuron before, after, and following wash of 4-AP. PPK1 current was evoked using a relief from inhibition protocol as described under “Experimental Procedures” (and Ref. ; noted with an arrowhead). Membrane potential was clamped to −60 mV with physiological solutions used for these experiments.

FIGURE 2.

4-AP decreases the activity of recombinant rat ASIC1a expressed in CHO cells in a dose-dependent manner. a, representative macroscopic current trace of recombinant rat ASIC1a expressed in a CHO cell before, after, and subsequent to washing 10 mm 4-AP with the acid stimulus (pH 5) added in the absence and presence of blocker at 2-min intervals. Cell membrane was clamped to −10 mV with physiological solutions used in these experiments. b, dose-response curve showing the effects of 4-AP (IC₅₀ = 0.76 ± 0.08 mm at −10 mV, n ≥ 7) on recombinant rat ASIC1a expressed in CHO cells. Data from experiments are similar to that shown in a.

FIGURE 3.

4-AP has differential effects on homo- and heteromeric acid-sensing ion channels. Overlays of representative traces (a) and corresponding summary graph (b) show the effects of 4-AP (10 mm at −10 mV) on macroscopic acid-sensing currents in CHO cells expressing recombinant ASIC1a, 1b, 2a, 3, and 1a plus 2a, and 1a plus 3. Activation in response to an acid stimulus (pH 5) provided in the absence and presence of 4-AP is shown with black and gray lines, respectively. At least four observations were made for each channel subtype. Physiologic bath and pipette solutions were used. *, significant decrease compared with before inhibitor when assessed with a paired t test. **, significantly less compared with the ASIC1a control group (ANOVA + Dunnett subtest).

FIGURE 4.

Differential effects of compounds related to 4-AP on recombinant ASIC1a. Representative traces (a) and corresponding summary graph (b, n ≥5) show the effects of vehicle (black) and pyridine and 4-MP (gray; at −10 mV) on macroscopic ASIC1a currents in CHO cells. ASIC1a was activated with pH 5. All compounds were used at 10 mm. Asymmetrical physiological solutions were used in these experiments. Neither pyridine nor 4-MP significantly affected the magnitude of macroscopic ASIC1a current.

FIGURE 5.

4-AP inhibits BLINaC. a, overlay of representative macroscopic current traces showing the effects of 1 mm 4-AP and 100 μm amiloride on recombinant rat BLINaC in CHO cells. cDNA encoding constitutively active BLINaC harboring the A443T mutation was used for these experiments. Inward current, as above, is downward. Current was evoked with a voltage ramp from 60 to −100 mV from a holding potential of 40 mV. Asymmetrical physiological solutions were used in these experiments. b, summary graph (n = 6) showing the effect of 4-AP (10 mm at −60 mV) on recombinant BLINaC in paired experiments. Data from experiments are similar to those in a. *, significant decrease compared with before 4-AP. c, dose-response curve showing the effects of 4-AP (at −60 mV) on recombinant BLINaC in CHO cells (IC₅₀ = 1.70 ± 0.18 mm, n = 5). Data from experiments are similar to those in a.

FIGURE 6.

4-AP inhibits BLINaC in a voltage-dependent manner. a, representative macroscopic current traces of rat BLINaC (A443T) in a voltage-clamped CHO cell before (left) and after (right) 10 mm 4-AP. Current was evoked with a 10-mV step protocol ranging from 100 to −100 mV from a holding potential of 0 mV. Symmetrical NaCl solutions were used in these experiments. b, summary graph of the macroscopic current-voltage (I-V) relation for recombinant rat BLINaC expressed in CHO cells in the absence (black) and presence (gray; 10 mm) of 4-AP. Data are from experiments identical to that in a (n = 5). c, corresponding conductance-voltage (G-V) curve for rat BLINaC in CHO cells in the presence of 10 mm 4-AP. Normalized conductance was fit with a Boltzmann distribution. d, summary graph showing the voltage dependence of the K_d for 10 mm 4-AP on BLINaC. The normalized (I_4-AP/I_con) I-V relation for BLINaC used to calculate K_d is shown in the inset. Data from experiments are identical to those in a.

FIGURE 7.

Voltage-sensitive changes in unitary conductance underpin the inhibitory actions of 4-AP on BLINaC. a, representative single-channel current traces of recombinant BLINaC (A443T) in excised, outside-out patches from CHO cells clamped to 60 (top) or −60 mV (bottom) before and after 5 μm amiloride and 10 mm 4-AP added in the presence of amiloride. Data are from continuous traces with representative segments shown for each condition. Breaks in the record are noted by solid vertical bars. Drugs were added to the extracellular bath solution. The open channel pore blocker, amiloride, was included with 4-AP at a submaximal dose (to increase channel “flicker”) in the latter part of the trace to facilitate calculation of unitary current (i) and conductance (g). Also for these experiments, the pipette and bath contained symmetrical LiCl solutions. Inward current is down, and the closed state (zero current level) is indicated with a dashed line and c. Summary graphs (n = 4) show the dose response of 4-AP on the single-channel activity (NP_o; b) and unitary conductance (g; c) of BLINaC at 60 (black) and −60 mV (gray). For these graphs, g and NP_o were normalized to that in the absence of 4-AP at the respective voltage. The inset in b shows the effect of submaximal amiloride on normalized NP_o at 60 (black) and −60 (gray) mV.

FIGURE 8.

4-AP decreases the unitary current of BLINaC in a dose-dependent manner. Representative segments from a typical (n = 4) continuous single-channel current trace of recombinant BLINaC (A443T) expressed in CHO cells in an excised, outside-out patch clamped to −60 mV before and after 4-AP in the absence and presence of amiloride. This patch contains at least two BLINaC channels. The pipette and bath contained symmetrical LiCl solutions. Inward current is down, and closed and open 1 and open 2 states are indicated with dashed lines and C, O₁, and O₂, respectively. The point at which amiloride was added is indicated by a solid gray arrowhead.