Acid-sensing ion channels in neurones of the rat suprachiasmatic nucleus

Abstract

We used reduced slice reparations to study ASIC-like currents in the rat central clock suprachiasmatic nucleus (SCN). In reduced SCN preparations, a drop of extracellular pH evoked a desensitizing inward current to excite SCN neurones to fire at higher rates. Under voltage-clamped conditions, all SCN neurones responded to a 5 s pH step to 6.4 with an inward current that decayed with an average time constant of 1.2 s to 10% of the peak at the end of step. The current was blocked by amiloride with an IC(50) of 14 microm and was carried mainly by Na(+), suggesting an origin of ASIC-like channels. The SCN neurones were sensitive to neutral pH, with 94% of cells responding to pH 7.0 with an inward current. The study of sensitivity to pH between 7.0 and 4.4 revealed a two-component dose-dependent H(+) activation in most SCN neurones, with the first component (85% in amplitude) having a pH(50) of 6.6, and the second (15%) a pH(50) of 5. The ASIC-like currents were potentiated by lactate and low Ca(2+), but were inhibited by Zn(2+). RT-PCR analysis demonstrated the presence of mRNA for ASIC1a, 2a, 2b, and 3 in SCN. Compared to other central neurones, the unique presence of ASIC3 along with ASIC1a in SCN neurones may contribute to the high pH sensitivity and unusual inhibition by Zn(2+). The high pH sensitivity suggests that the SCN neurones are susceptive to extracellular acidification of physiological origins and that the ASIC current might play a role in regulating SCN excitability.

Figure 1. Acid effects on SCN neurones

A and B, firing responses to the application of pH 6.4 solution of two representative cells recorded in the cell-attached (A) and the current-clamped (B) mode. Note the similar firing pattern, due to depolarisation block of spike generation, in both recording modes. C, desensitizing current response to the application of pH 6.4 solution of a representative cell voltage-clamped at a holding potential of −52 mV. The dashed line is the zero voltage level.

Figure 4. The ASIC-like current is carried mainly by Na⁺

A, both the ASIC-like current (upper left panel) and the voltage-activated Na⁺ current (upper right panel) reversed the direction of current flow at approximately the same potential (lower panel) in a representative cell. The ASIC-like current was activated by a 5 s pH step to 6.4 at holding potentials of −72, −52, −32, −12, −2, +8, +18, +28 and +48 mV (from bottom to top, respectively). The voltage-dependent Na⁺ current was activated by stepping to more depolarised potentials between −72 and +38 mV at 10 mV increments. Both currents had zero amplitude near +10 mV as indicated by the arrows. Note that the amplitude of ASIC-like current was magnified 10 times for better comparison. B, the averaged current–voltage relation indicated a reversal potential of ∼+13 mV, close to the predicted reversal potential of +19 mV for a Na⁺-selective channel. Each data point represents the mean ±s.e.m. of 7 cells.

Figure 2. Acid-evoked currents in SCN neurones

A, examples of acid-evoked currents from two representative SCN neurones in response to a 5 s pH step from 7.4 to 6.4. B and C, histograms showing the distribution of decay time constants (B) and I_5s/I_peak (the current amplitude at the end of 5 s pH step/peak amplitude) ratios (C). The biphasic neurones are included in the plots.

Figure 3. Amiloride blockade of the acid-evoked current

A, the acid-evoked current was reversibly blocked by 100 μm amiloride in a representative neurone. B, dose-dependent block by amiloride. The theoretical curve is calculated with the equation assuming a one-to-one binding of amiloride to and blockade of the channel, yielding an IC₅₀ value of 14 μm. Each data point represents the mean ±s.e.m. of at least 6 cells. C, amiloride reversibly blocked the acid response. Dashed lines are the zero voltage levels

Figure 5. pH dependency of the ASIC-like current

A, two representative SCN neurones showing two different pH-dependent activations of ASIC-like currents. The current amplitude in one cell (Aa, left panel) had saturated between pH 5.9 and 4.4 and could be accounted for by a one-component pH-dependent activation. The theoretical curve for the dose–response relation is calculated with a pH₅₀ of 6.67, assuming a three-to-one binding of H⁺ to and activation of the channel (Aa, right panel). The current amplitude in the second cell (Ab, left panel) levelled off between pH 5.9 and 5.4 but then doubled its amplitude at pH 4.4, and could only be described as a two-component pH-dependent activation. The theoretical curve for the dose–response relation is calculated with pH₅₀ of 6.6 and 5, with the amplitudes being 36% and 64%, respectively, for the first and second components (see Methods). A Hill coefficient of 3 was assigned for both components. All data points were obtained by normalizing to the peak current amplitude at pH 4.4. B, dose–response relation of the ASIC-like current amplitudes pooled from all 29 cells. The theoretical curve for the dose–response relation is calculated with pH₅₀ of 6.6 and 5, with the amplitudes being 85% and 15%, respectively, for the first and second components. Each data point represents the mean ±s.e.m. of 12 cells (Aa), 4 cells (Ab), and 25–29 cells (B). C, a representative cell showing the current response to pH 5.4, 4.4, 3.9, and 3.4, with current amplitude decreasing with lower pH (left panel). Right panel: superimposition of current traces after proper scaling to reveal a decrease in the decay time constant with lower pH. The scaling factor were 117%, 152%, and 217% for the current trace at pH 4.4, 3.9, and 3.4, respectively.

Figure 6. Modulation of the ASIC-like current by lactate (A), low Ca²⁺ (B), and Zn²⁺ (C)

A, 30 mm lactate potentiated the current response to pH 6.4 in a representative cell (left panel). Right panel: summary of experiments showing the potentiation by 30 mm lactate of the current response to pH 6.4 (n= 6). B, 0.5 mm Ca²⁺ potentiated the current response to pH 6.4 in a representative cell (left panel). Right panel: summary of experiments showing the potentiation by 0.5 mm Ca²⁺ of the current response to pH 6.4 (n= 6). C, 300 μm Zn²⁺ inhibited the current response to pH 5.4 in a representative cell (left panel). Right panel: summary of experiments showing the inhibition by 300 μm Zn²⁺ of current responses to pH 6.4 (n= 5), 5.4 (n= 14), and 4.4 (n= 2).

Figure 7. RT-PCR analysis of ASIC-encoding mRNA in SCN

RT-PCR was used to detect the expression of six ASIC subunits (1a, 1b, 2a, 2b, 3, and 4) in SCN. Positive controls were performed using cDNA from rat brain or DRG. The expected PCR product sizes for each ASIC subunit were 173, 234, 203, 212, 209, and 326 bp, respectively. Of note, a splice variant of ASIC3 transcript was found with expected size 541 bp. Negative controls were performed using RT products with omission of reverse transcriptase to examine the contamination of genomic DNA.