Modulation of Acid-sensing Ion Channel Currents, Acid-induced Increase of Intracellular Ca2+, and Acidosis-mediated Neuronal Injury by Intracellular pH

Abstract

Acid-sensing ion channels (ASICs), activated by lowering extracellular pH (pH o ), play an important role in normal synaptic transmission in brain and in the pathology of brain ischemia. Like pH o , intracellular pH (pH i ) changes dramatically in both physiological and pathological conditions. Although it is known that a drop in pH o activates the ASICs, it is not clear whether alterations of pH i have an effect on these channels. Here we demonstrate that the overall activities of ASICs, including channel activation, inactivation, and recovery from desensitization, are tightly regulated by pH i . In cultured mouse cortical neurons, bath perfusion of the intracellular alkalizing agent quinine increased the amplitude of the ASIC current by ϳ50%. In contrast, intracellular acidification by withdrawal of NH 4 Cl or perfusion of propionate inhibited the current. Increasing pH buffering capacity in the pipette solution with 40 mM HEPES attenuated the effects of quinine and NH 4 Cl. The effects of intracellular alkalizing/ acidifying agents were mimicked by using intracellular solutions with pH directly buffered at high/low values. Increasing pH i induced a shift in H ؉ dose-response curve toward less acidic pH but a shift in the steady state inactivation curve toward more acidic pH. In addition, alkalizing pH i induced an increase in the recovery rate of ASICs from desensitization. Consistent with its effect on the ASIC current, changing pH i has a significant influence on the acid-induced increase of intracellular Ca 2؉ , membrane depolarization, and acidosismediated neuronal injury. Our findings suggest that changes in pH i may play an important role in determining the overall function of ASICs in both physiological and pathological conditions. Acid-sensing ion channels (ASICs)3 are H ϩ -gated cation channels that belong to the Deg/ENaC superfamily (1). In peripheral sensory neurons, ASICs are implicated in nociception (2, 3), mechanosensation (4, 5), and taste transduction (6). In central neurons, ASICs play an important role in physiological processes such as synaptic transmission, learning, and memory (7-9). In pathological conditions including brain ischemia, ASICs are involved in acidosis-mediated glutamateindependent neuronal injury (10 -12), disclosing a novel therapeutic target for stroke intervention.The activities of ASICs are subjected to modulation by endogenous signaling molecules and biochemical changes associated with pathological conditions. For instance, Zn 2ϩ , an endogenous trace element released during neuronal activity, inhibits ASIC1a-containing channels with high affinity (13), whereas neurochemical components associated with tissue inflammation (e.g. Phe-Met-Arg-Phe (FMRF) amide) (14) and ischemia (e.g. lactate and arachidonic acid) (15-18) potentiate the ASIC currents. Delineating detailed modulation of ASICs by endogenous signaling molecules and biochemical changes is important for better understanding the exact and precise role these channels play in various physiological and pathological...

Identification of Protein Domains That Control Proton and Calcium Sensitivity of ASIC1a

“…Most modulators of ASIC1a function, including zinc, redox reagents, lactate, and PcTx1, act by altering the apparent proton sensitivity of the channel (16,18,25,(31)(32)(33)(34). These compounds also impact ASIC1a-induced neuronal death (8,9,15,31). We find that two protein regions, located between amino acids 87 and 197 and between amino acids 323-431 of the extracellular domain are both necessary and sufficient for high affinity proton sensing of ASIC1a.…”

“…Specifically, ASIC1a homomeric channels activate at pH values much closer to neutral pH compared with ASIC2a homomeric channels. The high affinity proton sensitivity of ASIC1a plays a prominent role in acidosisinduced neuronal death, and modulators that alter the pH dose response of ASIC1a affect neuronal sensitivity to prolonged acidosis (8,9,15). For example, the neuroprotective venom peptide PcTx1 increases the proton sensitivity of the ASIC1a channel, allowing the channel to desensitize at neutral pH and become unresponsive to subsequent acidic shifts in pH (16,17).…”

ASIC1a activation by amitriptyline and FMRF-amide is removed by serine proteases

“…This hypothesis is reinforced by the finding that usual therapeutic plasma concentrations of amitriptyline do not exceed 1 μM, 65 while the K D for inhibition of tetrodotoxin-sensitive Na + channels in rat dorsal root ganglia neurons is 4.7 μM. A consequence of the activatory effects of tricyclic antidepressants on ASIC1a channels, which have been shown to contribute to ischemia-related neuronal damage [20][21][22][23][24][25] is represented by changes in current therapy guidelines between long openings and adjacent brief closures) and decreased the proportion of mode 2 (association between brief openings and adjacent long closures).…”

“…17 ASIC-mediated cell Ca 2+ entry was correlated with a sustained proton-activated current component 8,9,18,19 and assumed to account for neurodegeneration consequent to ASIC activation in ischemic conditions. [20][21][22][23][24][25] Moreover, we have shown that ASIC1a Ca 2+ permeability is decreased by extracellular proteases, 26 presumably due to direct action at a cleavage site located in the extracellular loop of the channel. 27,28 Accurate single-channel recordings have reveled a wealth of details concerning the gating of ASICs: the existence of distinct gating modes and subconductance states, 29 stepwise activation by protonation of residues involved in Ca 2+ binding, 30 a long-lived desensitized state triggered by proton inflow.…”

Proton production, regulation and pathophysiological roles in the mammalian brain

“…Moreover, ASIC functions are reported to be regulated by intracellular pH [90] . These results indicate that intracellular protons can function as modulators.…”