Chai Sunghee, Li Minghua, Lan JingQuan, Xiong Zhi-Gang, Saugstad Julie A, Simon Roger P
Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, Oregon 97232, USA.
J Biol Chem. 2007 Aug 3;282(31):22668-77. doi: 10.1074/jbc.M703624200. Epub 2007 Jun 4.
Acid-sensing ion channel (ASIC) 1a and ASIC2a are acid-sensing ion channels in central and peripheral neurons. ASIC1a has been implicated in long-term potentiation of synaptic transmission and ischemic brain injury, whereas ASIC2a is involved in mechanosensation. Although the biological role and distribution of ASIC1a and ASIC2a subunits in brain have been well characterized, little is known about the intracellular regulation of these ion channels that modulates their function. Using pulldown assays and mass spectrometry, we have identified A kinase-anchoring protein (AKAP)150 and the protein phosphatase calcineurin as binding proteins to ASIC2a. Extended pulldown and co-immunoprecipitation assays showed that these regulatory proteins also interact with ASIC1a. Transfection of rat cortical neurons with constructs encoding green fluorescent protein- or hemagglutinin-tagged channels showed expression of ASIC1a and ASIC2a in punctate and clustering patterns in dendrites that co-localized with AKAP150. Inhibition of protein kinase A binding to AKAPs by Ht-31 peptide reduces ASIC currents in cortical neurons and Chinese hamster ovary cells, suggesting a role of AKAP150 in association with protein kinase A in ASIC function. We also demonstrated a regulatory function of calcineurin in ASIC1a and ASIC2a activity. Cyclosporin A, an inhibitor of calcineurin, increased ASIC currents in Chinese hamster ovary cells and in cortical neurons, suggesting that activity of ASICs is inhibited by calcineurin-dependent dephosphorylation. These data imply that ASIC down-regulation by calcineurin could play an important role under pathological conditions accompanying intracellular Ca(2+) overload and tissue acidosis to circumvent harmful activities mediated by these channels.
酸敏感离子通道（ASIC）1a和ASIC2a是中枢和外周神经元中的酸敏感离子通道。ASIC1a与突触传递的长期增强和缺血性脑损伤有关，而ASIC2a则参与机械感觉。尽管ASIC1a和ASIC2a亚基在大脑中的生物学作用和分布已得到充分表征，但对于调节这些离子通道功能的细胞内调节知之甚少。通过下拉实验和质谱分析，我们确定了A激酶锚定蛋白（AKAP）150和蛋白磷酸酶钙调神经磷酸酶为与ASIC2a结合的蛋白。扩展的下拉实验和免疫共沉淀实验表明，这些调节蛋白也与ASIC1a相互作用。用编码绿色荧光蛋白或血凝素标签通道的构建体转染大鼠皮质神经元，结果显示ASIC1a和ASIC2a在树突中呈点状和聚集状表达，且与AKAP150共定位。Ht-31肽抑制蛋白激酶A与AKAPs的结合会降低皮质神经元和中国仓鼠卵巢细胞中的ASIC电流，这表明AKAP150与蛋白激酶A结合在ASIC功能中发挥作用。我们还证明了钙调神经磷酸酶对ASIC1a和ASIC2a活性具有调节功能。钙调神经磷酸酶抑制剂环孢素A可增加中国仓鼠卵巢细胞和皮质神经元中的ASIC电流，这表明ASIC的活性受到钙调神经磷酸酶依赖性去磷酸化的抑制。这些数据表明，在伴有细胞内Ca(2+)过载和组织酸中毒的病理条件下，钙调神经磷酸酶对ASIC的下调可能在规避这些通道介导的有害活动中起重要作用。
