Acid-sensing ion channels in acidosis-induced injury of human brain neurons

Abstract

Acidosis is a common feature of the human brain during ischemic stroke and is known to cause neuronal injury. However, the mechanism underlying acidosis-mediated injury of the human brain remains elusive. We show that a decrease in the extracellular pH evoked inward currents characteristic of acid-sensing ion channels (ASICs) and increased intracellular Ca(2+) in cultured human cortical neurons. Acid-sensing ion channels in human cortical neurons show electrophysiological and pharmacological properties distinct from those in neurons of the rodent brain. Reverse transcriptase-PCR and western blot detected a high level of the ASIC1a subunit with little or no expression of other ASIC subunits. Treatment of human cortical neurons with acidic solution induced substantial cell injury, which was attenuated by the ASIC1a blockade. Thus, functional homomeric ASIC1a channels are predominantly expressed in neurons from the human brain. Activation of these channels has an important role in acidosis-mediated injury of human brain neurons.

Figure 1

Acid-sensing ion channel (ASIC) currents in human cortical neurons. (A and B) Phase-contrast image of human cortical neurons and example traces of TTX-sensitive Na⁺ current recorded in these neurons. (C) An example current trace showing N-methyl--aspartate (NMDA)-mediated response in human cortical neurons. (D) Example current traces showing γ-aminobutyric acid (GABA)-mediated response in human cortical neurons recorded at holding potentials of −40 and −60 mV. With CsF as the major ions in the recording electrode (see the section ‘Materials and methods'), the GABA-mediated currents are outward at −40 and −60 mV. (E and F) Representative traces and summary data showing the pH-dependent activation of ASIC currents in human cortical neurons. The dose–response curves were fit to Hill's equation with an average pH₅₀ of 6.60±0.02 (n=10). (G and H) Representative traces and summary data showing the current–voltage relationship in human cortical neurons. The acid-activated currents in human cortical neurons have a near linear I–V relationship with a reversal potential at ∼+60 mV. CsF, cesium fluoride.

Figure 2

Pharmacology of acid-sensing ion channels (ASICs) in human cortical neurons. (A and B) Representative current traces and summary data showing the blockade of ASIC currents by amiloride. (C and D) Representative current traces and summary data showing the inhibition of ASIC currents by PcTX1. (E and F) Representative current traces and summary data showing the potentiation of ASIC currents in human cortical neurons by the high-affinity zinc chelator TPEN. TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

Figure 3

Differential effects of high concentration of zinc on acid-sensing ion channel (ASIC) currents in human and mouse brain neurons. (A and B) Representative traces showing the effect of 150 μmol/L ZnCl₂ on ASIC currents in human (panel A) and mouse cortical neurons (panel B). (C) Summary data showing the effect of 150 μmol/L ZnCl₂ on the amplitude of ASIC currents in human and mouse cortical neurons. A volume of 150 μmol/L ZnCl₂ potentiates the ASIC current in mouse cortical neurons but inhibits the ASIC current in human cortical neurons (n=15 to 17).

Figure 4

Desensitization properties of acid-sensing ion channels (ASICs) in human cortical neurons. (A and B) Representative current traces and summary data showing pH-dependent desensitization of ASICs in human cortical neurons. (C and D) Representative traces and summary data showing a fast recovery of ASICs from desensitization in human cortical neurons. The time constant for the recovery of ASICs from desensitization is 0.9±0.23 secs (n=6). (E and F) Representative traces and summary data showing steady-state inactivation of ASIC currents in human cortical neurons. The pH₅₀ for steady-state inactivation of ASICs is 7.03±0.02 (n=7).

Figure 5

Acid-sensing ion channels (ASICs) in human cortical neurons are Ca²⁺ permeable. (A) Example traces and summary data showing acid-activated Ca²⁺ current in human cortical neurons at different membrane potentials. With Na⁺- and K⁺- free extracellular solution (ECF) containing 10 mmol/L Ca²⁺ as the only charge carrier, inward currents were recorded at pH 6.0. The average reversal potential is ∼−15 mV after correction of the liquid junction potential (n=5). (B) Representative images and summary data showing increase of [Ca²⁺]_i induced by a pH decrease from 7.4 to 6.0. Neurons were bathed in normal ECF containing 1.3 mmol/L CaCl₂ with blockers for both voltage-gated Ca²⁺ channels (e.g., 5 μmol/L nimodipine) and glutamate receptors (10 μmol/L MK-801 and 20 μmol/L CNQX). MK-801, (5S,10R)-(−)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione.

Figure 6

Acidosis induces injury of human cortical neurons through acid-sensing ion channel (ASIC)1a activation. (A) Phase-contrast image showing cultured human cortical neurons before and after incubation with either pH 7.4 or pH 6.0 solution. Twenty-four hours after 2 h incubation with pH 6.0 solution, loss of processes, swelling of the cell body, and positive propidium iodide (PI) staining can be seen (right panels). (B) Summary data showing the percentage of dead cells (PI positive) induced by acid incubation (n=31). Addition of ASIC blockers amiloride or PcTX1-inhibited acid-induced injury of human cortical neurons (n=79 and n=85). ^*P<0.05.

Figure 7

Biochemical and molecular biologic evidence supporting the presence of acid-sensing ion channels (ASICs) in human brain neurons. (A) Reverse transcriptase (RT)-PCR detection of rich expression of ASIC1a mRNA in human brain cortical tissue. In contrast to ASIC1a, only a weak expression of ASIC2a was seen and no expression of other known ASIC subunits were detected. (B) Western blot detection of strong ASIC1a and weak ASIC2a expression in human cortical tissues. (C) Immunohistochemical staining of human cortical neurons with ASIC1a antibody. DAPI (4′,6-diamidino-2-phenylindole) is used for nuclear staining.