Abstract:
Human acid-sensing ion channel 1b (hASIC1b) is a H(+)-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b\'s COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment.
摘要:
人酸敏感离子通道1b(hASIC1b)是H()门控阿米洛利敏感阳离子通道。我们先前已经表明神经胶质瘤细胞表现出阿米洛利敏感的阳离子电导。阿米洛利和ASIC1受体阻滞剂沙门毒素-1降低胶质瘤细胞的迁移和增殖。PKC还消除了神经胶质瘤细胞对阿米洛利敏感的电导,并抑制了平面脂质双层中的hASIC1b开放概率。此外,hASIC1b的COOH末端已显示与C激酶(PICK)1相互作用的蛋白质相互作用,将PKC靶向质膜。因此,我们检验了在特定PKC共有位点对hASIC1b的PKC调节抑制hASIC1b功能的假设。我们将hASIC1b中的三个共有PKC磷酸化位点(T26,S40和S499)突变为丙氨酸,为了防止磷酸化,谷氨酸或天冬氨酸,模拟磷酸化。我们的数据表明S40和S499是介导PKC调节hASIC1b的关键位点。我们在非洲爪狼卵母细胞中表达了突变的hASIC1b构建体,并通过双电极电压钳测量了酸激活电流。T26A和T26E没有表现出酸激活电流。S40A与野生型(WT)没有区别,而S40E,S499A,和S499D电流降低。PKC激活剂PMA和佛波醇12,13-二丁酸酯抑制WThASIC1b和S499A,在用PKC抑制剂白屈菜红碱预处理的卵母细胞中,PMA对S40A或WThASIC1b没有影响。白屈菜红碱抑制WThASIC1b和S40A,但对S499A或S40A/S499A无影响。PKC激活剂或抑制剂不影响WThASIC1b的表面表达。这些数据显示两个PKC共有位点S40和S499差异调节hASIC1b并介导PKC活化或PKC抑制对hASIC1b的作用。这将导致对神经胶质瘤细胞中PKC对该通道的调节有更深入的了解,这些信息可能有助于在治疗中设计潜在的有益疗法。