Regulation Of Acid-Sensing Ion Channel 1a(ASICla) Protein Expression And Function By Protein Kinase C

Protein phosphorylation is one of the most common post-translational modification of proteins.It is a universal mode of regulating life activities and plays an important role in both physiological and pathological conditions.Like many other proteins,membrane receptors and ion channels have become major targets for protein phosphorylation.Tissue acidosis is a common feature in many pathological conditions.Acid-sensing ion channels(ASICs)are highly expressed in peripheral sensory and central neurons and play a critical role in the perception of a wide range of p H changes in various conditions related to tissue acidosis.ASIC1 a is a major functional ASIC subunit in central neurons and a potential therapeutic target for neuronal injury,which activation plays a key role in acidosis-mediated neurotoxicity.Protein kinase C(PKC)activity or function has been proved to be associated with many physiological processes and pathological conditions,however,whether PKC activation regulates ASIC1 a protein expression and channel function remains ill defined.In this study,the effects of PKC phosphorylation on ASIC 1a protein expression and ASIC channel function were detected through Western blot,patch-clamp and cell migration analysis in primary cultured mouse cortical neurons,and in NS20 Y cells,a neuronal cell line,as well as in A172 cells,a human malignant glioma cell line.According to the pre-experimental results,cells were first incubated with phorbol-12-myristate-13-acetate(PMA,a PKC activator)at 200 n M.Compared with the control group,treatment with PMA for 6 h significantly increased ASIC1 a protein expression and ASIC current density in NS20 Y cells and in primary cultured mouse cortical neurons,while incubation with PMA for 24 h did not change ASIC1 a protein expression and ASIC current density.In contrast,treatment with Calphostin C(200 n M),a non-selective PKC inhibitor,for 6 h or longer decreased ASIC1 a protein expression and ASIC currents.Similar to Calphostin C,incubation with Go6976(1 M),a specific PKC and I inhibitor,for 24 h also reduced ASIC1 a protein expression,suggesting that PKC is involved in regulating ASIC1 a protein expression.Since activation of ASIC1 a plays an important role on acidosis-mediated cell injury,we explored the potential effect of PKC activator and inhibitor on acid-induced cell injury by measuring lactate dehydrogenase(LDH)release and MTT cell viability assay.We found that treatment of cells with PMA(200 n M)increased LDH release and aggravated acidosisinduced decrease in cell viability,while treatment of cells with Go6976(1 M)reduced LDH release and attenuated acidosis-induced decrease in cell viability in NS20 Y cells,which is consistent with their effects on ASIC1 a protein expression and channel function.In addition,we studied the effect of PKC activation on acid-mediated change of cell migration.We first confirmed that,comparing with p H7.4 extracellular fluid(ECF)incubation,weak acid(p H7.0 ECF)incubation could promote A172 cells migration in a monolayer scratch assay,and ASIC1 a specific inhibitor Pc Tx1(20 n M)could prevent this enhanced migration,confirming that activation of ASIC1 a channel is related to acid-induced cell migration.Next,A172 cells were incubated with PMA(200n M)or Go6976(1 M)respectively,followed by incubation with p H7.4 or p H7.0 ECF for 3 h.The data showed that incubation with PMA dramatically promoted the migration of A172 cells,while incubation with Go6976 significantly inhibited the migration of A172 cell at 24 h.Similarly,Pc Tx1 prevents the changes in cell migration,suggesting that PKC-induced change in cell migration is mediated,at least partially,by ASIC1 a.To examine the mechanism of PKC-mediated changes in ASIC1 a protein expression,a protein synthesis inhibitor Cycloheximide(CHX,20 M)was used to prevent new protein synthesis in NS20 Y cells.We found that the reduction in ASIC1 a protein expression by PKC inhibition involves a change in ASIC1 a protein degradation,which is mediated by ubiquitin proteasome system(UPS)-dependent degradation pathway,as MG-132(10 M),a proteasome inhibitor,can prevent Calphostin C-induced down-regulation of ASIC1 a protein expression.Last,we showed the evidence that PKC regulation of ASIC1 a protein expression involves NF-κB signaling pathway.A specific inhibitor IMD-0354(1 M),a nuclear factor kappa B(NF-κB)upstream inhibitor of kappa B kinase-(IKK-),was used to co-treated cells.Western blot analysis showed that PKC-mediated changes in ASIC1 a protein expression were totally inhibited in the presence of IMD-0354.Together,these results indicate that ASIC1 a protein expression and channel function are closely regulated by the activity of protein kinase C and its downstream signaling pathway(s).