Acid-sensitive ionic channels in midbrain dopamine neurons are sensitive to ammonium, which may contribute to hyperammonemia damage

Abstract

Acid-sensitive ion channels (ASICs) are proton-gated and belong to the family of degenerin channels. In the mammalian nervous system, ASICs are most well known in sensory neurons, where they are involved in nociception, occurring when injury or inflammation causes acidification. ASICs also are widely expressed in the CNS, and some synaptic roles have been revealed. Because neuronal activity can produce pH changes, ASICs may respond to local acidic transients and alter the excitability of neuronal circuits more widely than is presently appreciated. Furthermore, ASICs have been found to underlie calcium transients that contribute to neuronal death. Degeneration of midbrain dopamine neurons is characteristic of advanced idiopathic Parkinson's disease. Therefore, we tested for functional ASICs in midbrain dopamine neurons of the ventral tegmental area and substantia nigra compacta. Patch-clamp electrophysiology applied to murine midbrain slices revealed abundant acid-sensitive channels. The ASICs were gated and desensitized by extracellular application of millimolar concentrations of NH(4)Cl. Although the NH(4)Cl solution contains micromolar concentrations of NH(3) at pH 7.4, our evidence indicates that NH(4)(+) gates the ASICs. The proton-gated and the ammonium-gated currents were inhibited by tarantula venom (psalmotoxin), which is specific for the ASIC1a subtype. The results show that acid-sensitive channels are expressed in midbrain dopamine neurons and suggest that ammonium sensitivity is a widely distributed ASIC characteristic in the CNS, including the hippocampus. The ammonium sensitivity suggests a role for ASIC1s in hepatic encephalopathy, cirrhosis, and other neuronal disorders that are associated with hyperammonemia.

Fig. 1.

Proton-gated and ammonium-gated currents from mouse midbrain dopamine neurons. (A) Proton-gated currents were elicited by a 300-ms pressure application of acidified (pH 5.4) extracellular solution (open bar). Acid-activated current was not inhibited by bath application of a mixture of antagonists to glutamate, GABA, and acetylcholine ligand-gated ionic channels (gray bar). Tetrodotoxin was present in the extracellular solutions to inhibit sodium currents, and 50 mM tetraethylammonium was in the internal solution to inhibit I_h current. Hyperpolarizing pulses (−10 mV for 200 ms) applied before, after, and during the agonist application indicated that the proton-gated current arose from a 5.2 ± 0.2 (mean ± SE, n = 5) conductance increase over baseline. The conductance increase was estimated as the ratio of the conductance close to the maximum of the ligand-gated current to the conductance at the baseline (indicated by the arrows). Calibration bars represent 1 s and 200 pA for all current traces, which were all measured at a holding potential of −65 mV. (B) After 23 min to wash off the antagonists, the current was evoked by pressure application of an extracellular solution containing NH₄Cl (72 mM) at pH 7.4 (black bars). The current again was not inhibited by bath application of the non-ASIC antagonists (gray bar). The NH₄Cl-gated conductance increased 5.3 ± 0.3 (n = 4) times over baseline. (C) The dose–response dependence for the acid-gated current mediated by ASICs is shown. Data points were collected at 23°C (n = 4) by pressure-applying the pH-altered solutions with a puffer pipette. Thus, the exact pH hitting the cell surface was not the same as the pH in the puffer pipette, because the pressure-applied solution was diluted by the bath (see ref. 24).

Fig. 2.

Proton-gated and ammonium-gated currents are mediated by channels with common properties. (A) Proton-gated (pH 4.8, open bar) and NH₄Cl-gated (16 mM at pH 7.4, filled bar) currents were inhibited by P. cambridgei venom (1:1,000 dilution), an ASIC1a antagonist. (B) To test for cross-desensitization, currents activated by pressure-applied acidified solution (pH 4.8, open bar) were desensitized by bath-applied 8 mM NH₄Cl-containing solution. Likewise, currents elicited by 16 mM NH₄Cl-containing solution (filled bar) were desensitized by bath-applied solution with a pH of 6.8. Holding current was set to zero for comparison of the current traces. In A and B, the calibration bars represent 1 s and 200 pA for all current traces, and the holding potential was −65 mV. (C) To test for nonadditivity, pressure application of a solution at pH 4.8 (open bar, Left) activated a maximal current. Pressure application of 16 mM NH₄Cl to the same neuron at pH 7.4 evoked a smaller inward current (filled bar, Center). The current induced by the application of 16 mM NH₄Cl at pH 4.8 (open and filled bars) to the same neuron demonstrated no gain in amplitude (Right). Each trace is the average of three records. The calibration bars represent 1 s and 500 pA, and the holding potential was −65 mV.

Fig. 3.

Ammonium, not ammonia, gates the ASICs. Pressure application of NH₄Cl (16 mM) to a dopamine neuron at pH 7.5 (filled bar, Left) activated inward current. Pressure application of acidified solution (pH 7.1, open bar, Center) alone activated a very small current. Pressure application of 16 mM NH₄Cl at pH 7.1 (open and filled bars, Right), which increases the concentration of ammonium while decreasing the concentration of ammonia, to the same neuron activated a current with significantly larger amplitude. The holding potential was −65 mV. Each current trace is the average of three recordings.

Fig. 4.

Estimated minimum concentration for activation of ASICs by ammonium. (A) Example current traces are shown from the same cell for bath application (filled bar) of 4, 8, and 16 mM NH₄Cl. Calibration bars represent 5 min and 100 pA. (B) Example current traces are shown from the same cell upon pressure application by a puffer pipette containing NH₄Cl (filled bar) at the following concentrations: 16 mM (trace 1), 32 mM (trace 2), 64 mM (trace 3), 128 mM (trace 4), and 256 mM (trace 5). The actual concentration reaching the cell surface is lower than the puffer-pipette concentration. Calibration bars represent 1 s and 200 pA. (C) The initial part of the dose–response relationship is shown for bath-applied NH₄Cl. These currents give an accurate estimate of the lowest concentrations that activate detectable currents from dopamine neurons in this midbrain slice. Data points were averaged from 3 to 10 cells and represent the mean ± SE. (D) Dose–response dependence for ASIC activation by pressure-applied external NH₄Cl. The abscissa represents the concentration of NH₄Cl in the puffer pipette, but a lower concentration is reaching the cell surface. Data points were averaged from 3 to 11 cells and represent the mean ± SE. The curve (sigmoid logistic) indicates an apparent EC₅₀ value of 68 mM NH₄Cl (in the puffer pipette, not on the cell surface) and the apparent Hill coefficient of 1.6. Based on the currents evoked by bath-applied NH₄Cl, we may estimate that the actual EC₅₀ is closer to 20–40 mM ammonium.

Fig. 5.

Long-lasting depolarizations of dopamine neurons caused by hyperammonemia. (A) In voltage-clamp mode, bath-applied NH₄Cl (16 mM at pH 7.4, filled bar) induced transient and sustained inward holding current. Pressure application of extracellular acidified solution (pH 5.4; 300 ms every 60 s) induced transient currents, shown as brief downward deflections (inward current). These acid-induced currents are strongly desensitized by the bath-applied NH₄Cl. The holding potential was −65 mV. (B) In current-clamp mode, bath-applied NH₄Cl (16 mM at pH 7.4, filled bar) caused depolarization and evoked action potentials. (C) When P. cambridgei venom (1:1,000 dilution, gray bar), an ASIC1a antagonist, was applied during the current-clamp experiment, the depolarization was suppressed, and the spiking stopped. After wash-out of the venom, the depolarization and spiking resumed. During all of these experiments, glutamate receptors were inhibited by kynurenic acid (1 mM), GABAa receptors were inhibited by bicuculline (20 μM), and muscarinic receptors were inhibited by atropine (1 μM) in the extracellular solution. (Scale bars: 10 min.)