Dildy-Mayfield J E, Harris R A
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262, USA.
J Neurosci. 1995 Apr;15(4):3162-71. doi: 10.1523/JNEUROSCI.15-04-03162.1995.
Recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors expressed in oocytes are inhibited by ethanol and the sensitivity to ethanol depends on the kainate concentration and the subunit(s) expressed. For example, GluR3 kainate channels are more sensitive to inhibition by ethanol than GluR6 channels in the presence of maximally effective kainate concentrations. To determine if the ethanol inhibition was influenced by the cation permeability (Na+ vs Na+ and Ca2+) of the channels expressed, we compared ethanol inhibition of Ca(2+)-permeable glutamate receptors (GluRs) in oocytes perfused with normal- and high-Ca2+ buffers. The ethanol inhibition was much greater when Ca2+ was the only permeant cation. When Ba2+ was substituted for Ca2+, the ethanol inhibition was reduced, although it was still greater than with normal buffer. The enhanced ethanol inhibition of kainate-stimulated Ca2+ currents was reduced in oocytes injected with the Ca2+ chelator BAPTA, suggesting a role for intracellular Ca2+ in mediating enhanced ethanol sensitivity of kainate channels. The enhanced ethanol inhibition of Ca2+ currents was not due to a direct ethanol inhibition of Ca(2+)-stimulated Cl- currents in the oocyte because ethanol produced no effect on Ca(2+)-stimulated Cl-currents induced by injection of myo-inositol-1,4,5-trisphosphate. Because Ca2+ activates protein kinase C (PKC) and because we found that the PKC activator phorbol 12-myristate 13-acetate inhibits kainate responses (Dildy-Mayfield and Harris, 1994), we examined the role of PKC in mediating the enhanced ethanol inhibition of kainate responses produced by increased Ca2+. Inhibition of PKC by injection of the PKC inhibitor peptide or calphostin C prevented the enhanced ethanol inhibition of kainate-induced Ca2+ responses without altering ethanol inhibition in normal buffer. Thus, ethanol inhibition of kainate channels may involve two mechanisms, one that is independent of PKC and a second type that is due to activation of PKC under conditions of elevated Ca2+, resulting in enhanced inhibition of kainate responses.
卵母细胞中表达的重组α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）/海人藻酸受体受到乙醇的抑制，且对乙醇的敏感性取决于海人藻酸浓度和所表达的亚基。例如，在最大有效海人藻酸浓度存在的情况下，GluR3海人藻酸通道比GluR6通道对乙醇抑制更敏感。为了确定乙醇抑制是否受所表达通道的阳离子通透性（Na⁺ 与Na⁺ 和Ca²⁺）影响，我们比较了在灌注正常Ca²⁺ 缓冲液和高Ca²⁺ 缓冲液的卵母细胞中，乙醇对Ca²⁺ 通透型谷氨酸受体（GluRs）的抑制作用。当Ca²⁺ 是唯一的通透阳离子时，乙醇抑制作用更强。当用Ba²⁺ 替代Ca²⁺ 时，乙醇抑制作用减弱，尽管仍比正常缓冲液时更强。在注射Ca²⁺ 螯合剂BAPTA的卵母细胞中，海人藻酸刺激的Ca²⁺ 电流的乙醇增强抑制作用减弱，这表明细胞内Ca²⁺ 在介导海人藻酸通道乙醇敏感性增强中起作用。Ca²⁺ 电流的乙醇增强抑制作用并非由于乙醇直接抑制卵母细胞中Ca²⁺ 刺激的Cl⁻ 电流，因为乙醇对注射肌醇-1,4,5-三磷酸诱导的Ca²⁺ 刺激的Cl⁻ 电流没有影响。由于Ca²⁺ 激活蛋白激酶C（PKC），且我们发现PKC激活剂佛波酯12-肉豆蔻酸13-乙酸酯抑制海人藻酸反应（迪尔迪-梅菲尔德和哈里斯，1994年），我们研究了PKC在介导Ca²⁺ 增加所产生的海人藻酸反应的乙醇增强抑制中的作用。通过注射PKC抑制剂肽或钙泊三醇C抑制PKC可防止海人藻酸诱导的Ca²⁺ 反应的乙醇增强抑制，而不改变正常缓冲液中的乙醇抑制作用。因此，乙醇对海人藻酸通道的抑制可能涉及两种机制，一种独立于PKC，另一种是在Ca²⁺ 升高条件下由于PKC激活导致海人藻酸反应抑制增强。
