Studies On The Biological Properties Of Acid-sensing Ion Channels By RNA Interference

Acid-sensing ion channels (ASICs) are H⁺- gated cation channels expressed throughout mammalian central and peripheral nervous system. ASICs are activated by acidic pH and sodium enters the cells through this channel causing cell depolarization and excitation. They belong to the DEG/ENaC (degenerin/epithelial sodium channel) superfamily. Until now, six subunits of ASICs encoded by 4 genes have been cloned: ASIC1a, ASIC1b, ASIC2a, ASIC2b , ASIC3 and ASIC4. Among of them, ASIC1a is a most important element. It is associated with synaptic transmission and plasticity, spatial learning/memory, long-term potentiation(LTP), pain transduction and ischemia neuronal damage. It plays an important role in physiological and pathological conditions. Tissue acidification is a common pathological phenomenon in body, such as stroke, inflammation, ischemia, epilepsy, tumor tissue, metabolism abnormality and so on. It was demonstrated that acidosis played an important role in tissue injury. It was proved that ASIC1a was involved in neuronal damage. Activation of ASIC1a with resultant [Ca²⁺]i loading induces time-dependent neuronal injury occurring in the presence of the blockers for voltage-gated Ca²⁺ channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASIC1a, and by reducing [Ca²⁺]o. So our aim is to observe the possible neuroprotective effect by depressing the expression of ASIC1a with RNA interference.First we amplified the different subunits of ASICs in C6 cells (one kind of rat neuroglioma), rat hippocampal neurons and DRG neurons. Then RNAi technology was used to knock down the expression of ASIC1a and ASIC2a in C6 cells. The decreased level of ASICs was confirmed by western blotting. To study the effect of ASIC1a during acidosis-induced cell injury more conveniently, the stable ASIC1a-knock down C6 cells and negative control C6 cells were established after selection. The two kinds of cells were confirmed by semi-quantitative RT-PCR and western blotting.Through cell growth curve and colony formation experiments, we found that knock-down of ASIC1a had no influence on cell proliferation under normal pH condition. Then different extracellular pH values were set up to cause artificial cell injury. The pH values are 6.0, 6.5, 6.9, respectively. The acidosis-induced lesions to the two kinds of cells were observed by performing MTT assay and LDH release assay under different pH levels. It was shown that inhibiting the expression of ASIC1a can evidently decrease acidosis-induced injury and increase cell viability. Similar result was found on hippocampal neurons transfected with plasmid pshRNAla2.To explore if knock-down of ASIC1a is the main reason during resisting acidosis-induced damage, we observed the difference of adding amiloride, which is a non-selective blocker of ASICs channels or knock-down of ASIC1a in C6 cells. It was shown that the protective effect was almost the same under pH6.0 acidic condition. To study cellular and molecular mechanisms of acid-resistance in C6 cells, we first proved Ca²⁺ permeability through ASIC1a channel was existed. Then we observed the effects on shRNA1a2(2) and shNEG cells under pH6.0 acidic condition with [Ca²⁺ ]o rising from 0.2 mM to 1.3 mM. The results showed that the experiment group cells were involved in slight acid-induced neuronal injury comparing to control group cells.In conclusion, in this study, we found that inhibiting the expression of ASIC1a could significantly decrease acidosis-induced neuronal injury. Extracellular acidosis induced cell damage via ASIC1a-containing channels. The context-dependent activation of ASIC1a with acidification makes this channel a particularly intriguing target for new stroke therapies.