Acid sensing ion channels in dorsal spinal cord neurons

Abstract

Acid-sensing ion channels (ASICs) are broadly expressed in the CNS, including the spinal cord. However, very little is known about the properties of ASICs in spinal cord neurons compared with brain. We show here that ASIC1a and ASIC2a are the most abundant ASICs in mouse adult spinal cord and are coexpressed by most neurons throughout all the laminas. ASIC currents in cultured embryonic day 14 mouse dorsal spinal neurons mainly flow through homomeric ASIC1a (34% of neurons) and heteromeric ASIC1a plus 2a channels at a ratio of 2:1 (83% of neurons). ASIC2b only has a minor contribution to these currents. The two channel subtypes show different active pH ranges and different inactivation and reactivation kinetics supporting complementary functional properties. One striking property of native dorsal spinal neuron currents and recombinant currents is the pH dependence of the reactivation process. A light sustained acidosis induces a threefold slow-down of the homomeric ASIC1a (from pH 7.4 to pH 7.3) and heteromeric ASIC1a plus 2a (from pH 7.4 to pH 7.2) current reactivation (T(0.5) increasing from 5.77 to 16.84 s and from 0.98 to 3.2 s, respectively), whereas a larger acidosis to pH 6.6 induces a 32-fold slow-down of the ASIC1a plus 2a current reactivation (T(0.5) values increasing to 31.30 s). The pH dependence of ASIC channel reactivation is likely to modulate neuronal excitability associated with repetitive firing in response to extracellular pH oscillations, which can be induced, for example, by intense synaptic activity of central neurons.

Figure 1.

Pharmacological properties of ASIC currents recorded from mouse dorsal spinal neurons. A, Effect of PcTx1. Currents were recorded from wild-type neurons (All, type 1, type 2, type 3 neurons) and from ASIC2^−/− neurons, activated by pH drops from 7.4 to 5.0 each minute, at a holding potential of −50 mV. PcTx1 (20 nm) was applied between pH drops at pH 7.4. The current amplitude in the presence of PcTx1 (measured at the steady state of effect) was expressed as a ratio of the control current amplitude value (IPcTx1/I control) and plotted as mean ± SEM (n ranging from 12 to 47; ***p < 0.005 compared with type 1 current; ⁺p < 0.05, ⁺⁺⁺p < 0.005 compared with type 2 current). B, Original current traces recorded from three different neurons showing the effect of PcTx1 on a type 1 PcTx1-inhibited current (left), a type 2 PcTx1-resistant current (middle), and a type 3 half-inhibited current (right). For each neuron, a control current (○) and a current in the presence of 20 nm PcTx1 (●, steady-state effect) are shown. Currents were recorded at −50 mV and activated by a pH drop from 7.4 to 5 each minute. C, Effect of zinc. Currents were recorded from wild-type neurons (All, type 1, type 2, type 3 neurons) and from ASIC2^−/− neurons, activated by pH drops from 7.4 to 6.3 each minute, at a holding potential of −50 mV. ZnCl₂ (300 μm) was applied during pH drops. The current amplitude in the presence of zinc was expressed as a ratio of the control current amplitude value (I zinc/I control) and plotted as mean ± SEM (n ranging from 6 to 29; *p < 0.05, ***p < 0.005 compared with type 1 current; ⁺⁺p < 0.01, ⁺⁺⁺p < 0.005 compared with type 2 current). The dashed line figures the no-effect level. D, Original current traces recorded from three different neurons showing the effect of zinc (300 μm) on a type 1 current (left), a type 2 current (middle), and a type 3 current (right). For each neuron, a control current (○) and a current in the presence of 300 μm zinc (●) are shown. Currents were recorded at −50 mV and activated by a pH drop from 7.4 to 6.3 each minute.

Figure 2.

ASIC currents expressed by mouse dorsal spinal neurons mainly flow through ASIC1a and ASIC1a plus 2a channels. A, Inactivation time course. Currents were recorded from neurons (black bars, type 1, type 2, and ASIC2^−/−) and from transfected COS cells (white bars, ASIC1a, ASIC2a, and ASIC1a plus 2a), activated by pH drops (10 s) from 7.4 to 5.0, at a holding potential of −50 mV. The inactivation time constant τ was measured by an exponential fit of the current decay [I = A*exp(−t/τ) + C], and plotted as mean ± SEM (n ranging from 14 to 62; * p < 0.05, **p < 0.01, ***p < 0.005 compared with ASIC1a current). B, pH-dependent activation. Currents were recorded from neurons (black symbols: ▲, type 1; ●, type 2; ♦, All; ■, ASIC2^−/−) and from transfected COS cells (white symbols: △, ASIC1a; □, ASIC2a; ○, ASIC1a plus 2a), activated by pH drops (10 s) from 7.4 to a variable pH test, at a holding potential of −50 mV. The current amplitude was expressed as a ratio of the amplitude of the current elicited by a pH drop from 7.4 to 5.0 (I/I control) and plotted as mean ± SEM (n ranging from 3 to 73) as a function of the pH test. Data were fitted as a dose–response sigmoidal curve following the equation Y = Y_min + [(Y_max − Y_min)/(1 + 10̂[Log (pH _0.5 − X)*n_H])], where pH_0.5 is pH of half-maximal activation, n_H is Hill number, Y_min and Y_max were 0 and 1, respectively, except for ASIC2a (□), where Y_max was 16 (incomplete curve shown). ▲, pH_0.5 = 6.46, n_H = 1.50; ■, pH_0.5 = 6.51, n_H = 1.39; △, pH_0.5 = 6.37, n_H = 1.25; ♦, pH_0.5 = 6.12, n_H = 1.39; ●, pH_0.5 = 6.03, n_H = 1.94; ○, pH_0.5 = 6.06, n_H = 2.15; □, pH_0.5 = 3.87, n_H = 1.05. C, pH-dependent inactivation. Currents were recorded from neurons (black symbols: ▲, type 1; ●, type 2; ■, ASIC2^−/−) and from transfected COS cells (white symbols: △, ASIC1a; □, ASIC2a; ○, ASIC1a plus 2a), activated by pH drops (10 s) from a pH test to 5.0, at a holding potential of −50 mV. The current amplitude was expressed as a ratio of the amplitude of the current elicited by a pH drop from pH 7.4 to 5.0 (I/I control) and plotted as mean ± SEM (n ranging from 4 to 24) as a function of the pH test. Data were fitted as a dose–response sigmoidal curve following the equation Y = Y_min + [(Y_max − Y_min)/(1 + 10̂[Log (pH _0.5 − X)*n_H])], where pH_0.5 is pH of half-maximal activation, n_H is Hill number. ▲, ■, △, pH_0.5 = 7.30, n_H = 4.6, Y_max = 1.3; ●, pH_0.5 = 6.74, n_H = 3.82; ○, pH_0.5 = 6.46; n_H = 3.82; □, pH_0.5 = 6.16, n_H = 3.82.

Figure 3.

Reactivation time course of ASIC currents expressed by mouse dorsal spinal neurons. A, Currents (▲, type 1; ●, type 2; ■, ASIC2^−/−) were activated by repetitive pH drops from pH 7.4 to 5.0 at a holding potential of −50 mV. The current amplitude was expressed as a ratio of the amplitude of the control current (I/I control) and plotted as mean ± SEM (n ranging from 3 to 72) as a function of the time interval (s) between the end of the control pH drop and the onset of the next pH drop. Data were fitted as a one-phase exponential following the equation Y = Ymax*[1 − exp(−k*t)], with half reactivation time T_0.5 = 0.69/k. ▲, T_0.5 = 3.59 s; ■, T_0.5 = 5.49 s; ●, T_0.5 = 0.98 s. Inset, Enlargement on the first 10 s. B, Original current traces recorded from three dorsal spinal neurons illustrating the difference of reactivation time courses between a type 1 current (top), an ASIC2^−/− current (middle), and a type 2 current (bottom). For each neuron, several traces are superimposed, showing one control current and several subsequent reactivated currents recorded after different time intervals (s) from the end of the control pH drop: 2, 4, 8, 10, 18, and 20 s for type 1 current; 2, 4, 6, 16, and 22 s for ASIC2^−/− current; and 0.5, 1, 2, 4, and 8 s for type 2 current. Currents were recorded at −50 mV and activated by repetitive pH drops from pH 7.4 to 5.0. Only the first pH drop corresponding to the control current is figured by a black line.

Figure 4.

Modulation of the reactivation time course of dorsal spinal neuron type 2 current by extracellular pH, and consequences on AP triggering. A, pH-dependent reactivation of type 2 current. Currents were recorded at a holding potential of −50 mV and activated by pH drops from various resting pH values to pH 5.0. The current amplitude was expressed as a ratio of the amplitude of the control current (I/I control) and plotted as mean ± SEM (n ranging from 3 to 72) as a function of the time interval (s) between the end of the control pH drop and the onset of the next pH drop. Data were fitted as a one-phase exponential following the equation: Y = Ymax*[1 − exp(−k*t)], with half-reactivation time T_0.5 = 0.69/k. ○, pH 9.0, T_0.5 = 0.44 s; ●, pH 7.4, T_0.5 = 0.98 s; △, pH 7.2, T_0.5 = 3.21 s; ▲, pH 6.9, T_0.5 = 8.13 s; □, pH 6.7, T_0.5 = 17.03 s; ■, pH 6.6, T_0.5 = 31.27 s. Inset, Enlargement on the first 10 s. B, Original current traces recorded from one single neuron. Currents were recorded at −50 mV and activated by pH drops to pH 5.0 from pH 7.4 (top), pH 7.2 (middle), and pH 6.9 (bottom). For each resting pH value, several traces are superimposed, showing one control current and several subsequent reactivated currents recorded after different time intervals (s) from the end of the control pH drop: 1, 2, 3, and 10 s for pH 7.4; 2, 3, 4, 5, 6, 7, 10, 13, and 15 s for pH 7.2; and 2, 3, 4, 5, 6, 10, and 20 s for pH 6.9. Only the first pH drop corresponding to the control current is figured by a black line. C, pH-dependent reactivation of type 2 current-induced depolarization. Depolarizations were recorded in current-clamp mode from a mean membrane potential of −51.54 ± 1.1 mV and activated by pH drops from various resting pH values to pH 5.0 or pH 6.0. The depolarization amplitude was expressed as a ratio of the control depolarization (dV/dV control) and plotted as mean ± SEM (n ranging from 3 to 18) as a function of the time interval (s) between the end of the control pH drop and the onset of the next pH drop. Data were fitted as a one-phase exponential following the equation: Y = Ymax*[1 − exp(−k*t)], with half reactivation time T_0.5 = 0.69/k. ○, pH 9.0, T_0.5 = 0.03 s; ●, pH 7.4, T_0.5 = 0.17 s; △, pH 7.2, T_0.5 = 0.95 s; ▲, pH 6.9, T_0.5 = 2.61 s; □, pH 6.7, T_0.5 = 6.06 s. Inset, Enlargement on the first 10 s. D, Original type 2 current and potential traces recorded from one single dorsal spinal neuron. Depolarizations were activated by pH drops to pH 6.0 from pH 7.4 (top), pH 7.2 (middle), and pH 6.9 (bottom). For each resting pH value, several traces are superimposed, showing one control depolarization and several subsequent reactivated depolarizations recorded after different time intervals (s) from the end of the control pH drop: 0.1, 0.3, and 2 s at pH 7.4; 0.4, 0.7, 1, 2, and 4 s at pH 7.2; and 1, 2, 3, 5, and 10 s at pH 6.9. Only the first pH drop corresponding to the control depolarization is figured by a black line. The 0 mV level is figured by a dashed line. Inset, Superimposed currents traces recorded at −50 mV and activated by pH drops from pH 7.4 to pH 6.0 after different time intervals (1, 2, 3 s) from the end of the control (first) pH drop.

Figure 5.

Modulation of the reactivation time course of dorsal spinal neuron ASIC2^−/− current by extracellular pH and consequences on AP triggering. A, pH-dependent reactivation of ASIC2^−/− current. Currents were recorded at a holding potential of −50 mV and activated by pH drops from various resting pH values to pH 5.0. The current amplitude was expressed as a ratio of the amplitude of the control current (I/I control) and plotted as mean ± SEM (n ranging from 3 to 20) as a function of the time interval (s) between the end of the control pH drop and the onset of the next pH drop. Data were fitted as a one-phase exponential following the equation: Y = Ymax*[1 − exp(−k*t)], with half reactivation time T_0.5 = 0.69/k. ○, pH 9.0, T_0.5 = 3.3 s; ●, pH 7.4, T_0.5 = 5.77 s; ▽, pH 7.3, T_0.5 = 16.84 s. Inset, Enlargement on the first 10 s. B, Original current traces recorded from one single ASIC2^−/− neuron. Currents were recorded at −50 mV and activated by pH drops to pH 5.0 from pH 7.4 (top) and pH 7.3 (bottom). For each resting pH value, several traces are superimposed, showing one control current and several subsequent reactivated currents recorded after different time intervals (s) from the end of the control pH drop: 1, 2, 4, 10, and 15 s for pH 7.4; and 2, 5, 10, and 20 s for pH 7.3. Only the first pH drop corresponding to the control current is figured by a black line. C, pH-dependent reactivation of ASIC2^−/− current-induced depolarization. Depolarizations were recorded in current-clamp mode and activated by pH drops from various resting pH values to pH 6.0. The depolarization amplitude was expressed as a ratio of the control depolarization (dV/dV control) and plotted as mean ± SEM (n ranging from 3 to 18) as a function of the time interval (s) between the end of the control pH drop and the onset of the next pH drop. Data were fitted as a one-phase exponential following the equation: Y = Ymax*[1 − exp(−k*t)], with half reactivation time T_0.5 = 0.69/k. ○, pH 9.0, T_0.5 = 0.46 s; ●, pH 7.4, T_0.5 = 2.24 s; ▽, pH 7.3, T_0.5 = 7.19 s; △, pH 7.2, T_0.5 = 12.55 s. Inset, Enlargement on the first 10 s. D, Original potential traces recorded from one single dorsal spinal ASIC2^−/− neuron. Depolarizations were activated by pH drops to pH 6.0 from pH 7.4 (top) and pH 7.3 (bottom). For each resting pH value, several traces are superimposed, showing one control depolarization and several subsequent reactivated depolarizations recorded after different time intervals (s) from the end of the control pH drop: 0.5, 2, 10, and 15 s at pH 7.4; and 2, 6, 10, 15, 20, and 25 s at pH 7.3. Only the first pH drop corresponding to the control depolarization is figured by a black line. The 0 mV level is figured by a dashed line.

Figure 6.

Relative expression of ASIC mRNAs in mouse spinal cord. Quantitative RT-PCR realized on total RNA extracts from adult spinal cord (Adult), fetal E14 spinal cord (E14), fetal E14 dorsal spinal cord (E14 dorsal), and primary cultured neurons from fetal E14 dorsal spinal cord after 2 (J2), 7 (J7), 13 (J13), and 21 (J21) days of culture. Expression of each ASIC is expressed as a ratio of the expression of ASIC1a in the same extract.

Figure 7.

Localization of ASIC1a and ASIC2a in mouse adult spinal cord. A, In situ hybridization of ASIC1a (a) and ASIC2a (b) on adult spinal cord sections, detected using DAB with a light cresyl violet counter-staining. For each ASIC, a hemi-spinal cord is shown (scale bar, 200 μm) along with three enlargements (scale bar, 100 μm) localized by black boxes on the corresponding hemi-spinal cord. DAB-labeled cells appear in brown to black (red arrow), and DAB-unlabeled cells appear in pink to violet (blue arrow). Orientation of the slices and position of one to nine laminas are figured on a drawing (c) of the spinal cord at the lumbar 5 level. B, Immunohistolabeling of ASIC2a (green) was performed on the same adult spinal cord section than in situ hybridization for ASIC1a detected by immunofluorescence (red). Labeling for ASIC1a (a, d, g), ASIC2a (b, e, h), and the merged image (c, f, i) is shown in lamina 2 (a–c), lamina 5 (d–f), and lamina 9 (g–i) of the same spinal cord. White arrows indicate examples of double-labeled neurons. Scale bar, 20 μm.