ASIC-like, proton-activated currents in rat hippocampal neurons

Abstract

The expression of mRNA for acid sensing ion channels (ASIC) subunits ASIC1a, ASIC2a and ASIC2b has been reported in hippocampal neurons, but the presence of functional hippocampal ASIC channels was never assessed. We report here the first characterization of ASIC-like currents in rat hippocampal neurons in primary culture. An extracellular pH drop induces a transient Na(+) current followed by a sustained non-selective cation current. This current is highly sensitive to pH with an activation threshold around pH 6.9 and a pH(0.5) of 6.2. About half of the total peak current is inhibited by the spider toxin PcTX1, which is specific for homomeric ASIC1a channels. The remaining PcTX1-resistant ASIC-like current is increased by 300 microM Zn(2+) and, whereas not fully activated at pH 5, it shows a pH(0.5) of 6.0 between pH 7.4 and 5. We have previously shown that Zn(2+) is a co-activator of ASIC2a-containing channels. Thus, the hippocampal transient ASIC-like current appears to be generated by a mixture of homomeric ASIC1a channels and ASIC2a-containing channels, probably heteromeric ASIC1a+2a channels. The sustained non-selective current suggests the involvement of ASIC2b-containing heteromeric channels. Activation of the hippocampal ASIC-like current by a pH drop to 6.9 or 6.6 induces a transient depolarization which itself triggers an initial action potential (AP) followed by a sustained depolarization and trains of APs. Zn(2+) increases the acid sensitivity of ASIC channels, and consequently neuronal excitability. It is probably an important co-activator of ASIC channels in the central nervous system.

Figure 1. ASIC-like current in hippocampal neurons

A, hippocampal ASIC-like currents activated at pH 5 and recorded at −60, −30, +10, +30 and +60 mV. The 0 pA current level is indicated on the left. B, current-potential relationship of hippocampal ASIC-like current obtained from traces shown in A. ○, peak current; •, plateau current. C, hippocampal ASIC-like currents induced by pH 6.9, 6 and 5. Currents were recorded at −50 mV. D, pH-dependent activation of the hippocampal ASIC-like current. Current amplitude was expressed as a fraction of the current induced by pH 5 (I/I_{pH 5}), and plotted as mean ± s.e.m., n ranging from 8 to 18. Between pH 7.4 and 5, data could be fitted by a sigmoidal curve, showing a pH_0.5 of 6.2 and a Hill coefficient of 1.48. The dashed curves represent the pH-dependent activation of the homomeric ASIC1a and of the heteromeric ASIC1a+2a (1:1) currents expressed in Xenopus oocytes (Baron et al. 2001).

Figure 2. Pharmacological properties of the hippocampal ASIC-like current

A, reversible inhibition of hippocampal ASIC-like current by 500 μm amiloride. B, reversible inhibition of hippocampal ASIC-like current by 10 nm of the toxin PcTX1. Currents were recorded at −50 mV. Amiloride and PcTX1 were given before and during the pH drop.

Figure 3. Effect of Zn²⁺ on the hippocampal ASIC-like current

A, 300 μm Zn²⁺ co-applied with acidic pH increases the amplitude of the peak hippocampal ASIC-like current without affecting the plateau phase. Currents were recorded at −50 mV. B, effect of 300 μm Zn²⁺ on hippocampal ASIC-like currents induced by pH ranging from 6.9 to 5. Current amplitude ratio (I_Zn/I_control) was measured for each pH value, in the absence (□) and in the presence of 10 nm PcTX1 (▪), and plotted as mean ± s.e.m., n ranging from 4 to 22; * significantly different from corresponding pH 5 ratio (P < 0.05); † Significantly different from corresponding control (no PcTX1) pH ratio (P < 0.005). Currents were recorded at −50 mV. On the right side on the histogram, original current traces are shown to illustrate the effect of Zn²⁺ on hippocampal ASIC-like current induced by pH 6.9 (top) and pH 6 (bottom) in the presence of PcTX1 (holding potential: −50 mV). C, pH-dependent activation of the PcTX1-sensitive Zn²⁺-insensitive ASIC-like current (•) and of the PcTX1-resistant Zn²⁺-sensitive ASIC-like current (○). Data for PcTX1-resistant ASIC-like current were obtained from the same experiments as in B. Data for PcTX1-sensitive Zn²⁺-insensitive ASIC-like current were obtained from neurons (20 % of total recorded neurons) showing an ASIC-like current highly sensitive to PcTX1 (90–100 % inhibited) and not potentiated by Zn²⁺. Current amplitude was expressed as a fraction of the current induced by pH 5 (I/I_pH5), and plotted as mean ± s.e.m., n ranging from 4 to 18. Between pH 7.4 and 5, data could be fitted by sigmoidal curves, showing a pH_0.5 of 6.3 for the PcTX1-sensitive Zn²⁺-insensitive ASIC-like current and a pH_0.5 of 6.0 for the PcTX1-resistant Zn²⁺-sensitive ASIC-like current.

Figure 4. Effect of ASIC current activation on membrane potential of hippocampal neurons

A, membrane depolarization induced by pH 6.9 (a), pH 6.6 (b) and pH 5 (c) in a single neuron. The initial acid-induced depolarization and AP is shown on a higher scale (100-fold) under each recording. Membrane potential was recorded in current-clamp mode with 0 pA current. Resting potential was −63 mV. Ticks on the left side of recordings represent the 0 mV level (upper tick) and the −60 mV level (lower tick). On the same neuron, ASIC-like currents activated by pH 6.6 and 5 were subsequently recorded in voltage-clamp mode at −60 mV (d). B, mean maximal membrane depolarization induced by ASIC-like current activation (V_max −V_rest) as a function of extracellular pH. Recordings with a resting membrane potential (V_rest) between −45 and −55 mV were selected, and action potentials were excluded of measurements. Mean ± s.e.m. values are shown, n ranging from 3 to 12. C, mean maximal membrane depolarization induced by ASIC-like current activation (V_max −V_rest) as a function of current density. For membrane potential measurements, recordings with a resting membrane potential between −45 and −55 mV (V_rest) were selected, and action potentials were excluded of measurements. ASIC-like current density (pA pF⁻¹) was subsequently measured on the same neurons, at holding potential between −45 and −55 mV. Membrane potential and ASIC-like current amplitude were measured during the transient phase (•) and during the sustained plateau phase (○) for the same pH value. Mean ± s.e.m. values are shown, n ranging from 3 to 12. D, effect of 300 μm Zn²⁺ on membrane depolarization induced by ASIC-like current activation. Membrane depolarization induced by pH 6.9 (a) and pH 6.9 + 300 μm Zn²⁺ (b) on a single neuron. Membrane potential was recorded in current-clamp mode with 0 pA current. Resting membrane potential was −53 mV. Ticks on the left side of recordings represent the 0 mV level (upper tick) and the −60 mV level (lower tick).