González Marco I, Kazanietz Marcelo G, Robinson Michael B
Department of Pediatrics and Pharmacology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Mol Pharmacol. 2002 Oct;62(4):901-10. doi: 10.1124/mol.62.4.901.
In previous studies, we have shown that activation of protein kinase C (PKC) rapidly (within minutes) increases the activity and cell surface expression of the glutamate transporter EAAC1 in two systems that endogenously express this transporter (C6 glioma cells and cocultures of neurons and astrocytes). However, the magnitude of the increase in activity is greater than the increase in cell surface expression. In addition, certain compounds completely block the increase in cell surface expression but only partially attenuate the increase in activity. We hypothesized that PKC increases EAAC1 activity by increasing cell surface expression and catalytic efficiency and that two different subtypes of PKC mediate these effects. To address these hypotheses, the PKC subtypes expressed by C6 glioma cells were identified. Of the PKC subtypes that are activated by phorbol esters, only PKCalpha, PKCdelta, and PKCepsilon were observed. Gö6976, a compound that blocks PKCalpha at concentrations that do not inhibit PKCdelta or PKCepsilon, partially inhibited the increase in uptake but completely abolished the increase in EAAC1 cell surface expression. The 'Gö6976-insensitive' increase in activity was not associated with a change in total transporter expression but was associated with an increase in the V(max). Na(+)-dependent glycine transport was not increased, providing indirect evidence that the Gö6976-insensitive increase in activity was not caused by a change in the Na(+) electrochemical gradient required for activity. Finally, by down-regulating different subtypes of PKC, we found evidence that PKCepsilon mediates the increase in EAAC1 activity that is independent of changes in cell surface expression and found further evidence that PKCalpha mediates the increase in cell surface expression. The potential relationship of the present work with a previously identified role for PKCalpha in certain forms of synaptic plasticity is discussed.
在先前的研究中，我们已经表明，在两个内源性表达谷氨酸转运体EAAC1的系统（C6胶质瘤细胞以及神经元与星形胶质细胞的共培养物）中，蛋白激酶C（PKC）的激活会迅速（在数分钟内）增加谷氨酸转运体EAAC1的活性及细胞表面表达。然而，活性增加的幅度大于细胞表面表达增加的幅度。此外，某些化合物能完全阻断细胞表面表达的增加，但仅部分减弱活性的增加。我们推测，PKC通过增加细胞表面表达和催化效率来提高EAAC1的活性，并且两种不同的PKC亚型介导这些效应。为了验证这些推测，我们鉴定了C6胶质瘤细胞表达的PKC亚型。在被佛波酯激活的PKC亚型中，仅观察到PKCα、PKCδ和PKCε。Gö6976是一种在不抑制PKCδ或PKCε的浓度下阻断PKCα的化合物，它部分抑制了摄取的增加，但完全消除了EAAC1细胞表面表达的增加。活性的“Gö6976不敏感”增加与转运体总表达的变化无关，但与V(max)的增加有关。Na⁺依赖性甘氨酸转运未增加，这间接证明了Gö6976不敏感的活性增加不是由活性所需的Na⁺电化学梯度变化引起的。最后，通过下调不同的PKC亚型，我们发现有证据表明PKCε介导了与细胞表面表达变化无关的EAAC1活性增加，并且进一步发现证据表明PKCα介导了细胞表面表达的增加。本文还讨论了当前工作与先前确定的PKCα在某些形式的突触可塑性中的作用之间的潜在关系。
