Hawrysh Peter John, Miles Ashley Rebecca, Buck Leslie Thomas
Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.
Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada; Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.
Comp Biochem Physiol B Biochem Mol Biol. 2016 Oct;200:44-53. doi: 10.1016/j.cbpb.2016.06.002. Epub 2016 Jun 6.
Neurons from the western painted turtle (Chrysemys picta bellii) are remarkably resilient to anoxia. This is partly due to a reduction in the permeability of excitatory glutamatergic ion channels, initiated by mitochondrial ATP-sensitive K(+) (mK(+)ATP) channel activation. The aim of this study was to determine if: 1) PKCε, a kinase associated with hypoxic stress tolerance, is more highly expressed in turtle brain than the anoxia-intolerant rat brain; 2) PKCε translocates to the mitochondrial membrane during anoxia; 3) PKCε modulates mK(+)ATP channels at the Thr-224 phosphorylation site on the Kir6.2 subunit; and 4) Thr-224 phosphorylation sensitises mK(+)ATP channels to anoxia. Soluble and mitochondrial-rich particulate fractions of turtle and rat cerebral cortex were isolated and PKCε expression was determined by Western blot, which revealed that turtle cortical PKCε expression was half that of the rat. Following the transition to anoxia, no changes in PKCε expression in either the soluble or particulate fraction of the turtle cortex were observed. Furthermore, incubation of tissue with tat-conjugated activator or inhibitor peptides had no effect on the amount of PKCε in either fraction. However, we observed a 2-fold increase in Thr-224 phosphorylation following 1h of anoxia. The increased Thr-224 phosphorylation was blocked by the general kinase inhibitor staurosporine but this did not affect the latency or magnitude of mK(+)ATP channel-mediated mitochondrial depolarization following anoxia, as indicated by rhodamine-123. We conclude that PKCε does not play a role in the onset of mitochondrial depolarization and therefore glutamatergic channel arrest in turtle cerebral cortex.
西部锦龟（Chrysemys picta bellii）的神经元对缺氧具有显著的耐受性。这部分归因于线粒体ATP敏感性钾离子（mK(+)ATP）通道激活引发的兴奋性谷氨酸能离子通道通透性降低。本研究的目的是确定：1）与低氧应激耐受性相关的蛋白激酶Cε（PKCε）在龟脑中的表达是否高于不耐缺氧的大鼠脑；2）缺氧期间PKCε是否转位至线粒体膜；3）PKCε是否在Kir6.2亚基的苏氨酸-224磷酸化位点调节mK(+)ATP通道；4）苏氨酸-224磷酸化是否使mK(+)ATP通道对缺氧敏感。分离了龟和大鼠大脑皮层的可溶性和富含线粒体的颗粒部分，通过蛋白质免疫印迹法测定PKCε的表达，结果显示龟皮层PKCε的表达是大鼠的一半。在转为缺氧状态后，未观察到龟皮层可溶性或颗粒部分中PKCε表达的变化。此外，用tat偶联的激活剂或抑制剂肽孵育组织对任一部位的PKCε含量均无影响。然而，我们观察到缺氧1小时后苏氨酸-224磷酸化增加了2倍。一般激酶抑制剂星形孢菌素可阻断苏氨酸-224磷酸化的增加，但这并不影响缺氧后mK(+)ATP通道介导的线粒体去极化的潜伏期或幅度，如罗丹明-123所示。我们得出结论，PKCε在龟脑皮层线粒体去极化的起始过程中不起作用，因此也不参与谷氨酸能通道阻滞。
