Identification of signal transduction cascades involved in long-lasting potentiation of inhibitory synapses

在小脑浦肯野细胞，GABA能抑制性突触传递在兴奋性攀爬纤维输入激活后经历了持久的“反弹增强”。反弹增强是由攀爬纤维引起的细胞内Ca^2+浓度的短暂升高触发的，并表现为突触后GABA_A受体敏感性的持久增加。在本系列研究中，我发现Ca^2+/钙调蛋白依赖性激酶II（CaM-KII）信号转导通路的抑制剂有效地阻断了反跳增强的诱导。一种非选择性蛋白激酶抑制剂，星形孢菌素，有效地阻止了诱导反弹增强。钙调素拮抗剂calmidazolium和CaM-KII拮抗剂KN-62在Ca^2+通道激活前和激活过程中细胞外应用时，可有效阻断反跳增强。另一方面，这些拮抗剂对一旦建立的反弹增强没有影响。 ...更多信息 细胞内应用的肽拮抗剂的CaM-KII也阻止了诱导反弹增强。这些抑制剂对电压门控Ca^2+通道电流或基础抑制传递本身没有影响。此外，细胞内注射活性CaM-KII和应用磷酸酶抑制剂calyculin A，诱导GABA_A受体介导的电流增强。这些结果强烈表明，RP涉及CaM-KII介导的蛋白磷酸化。GABA_A受体的功能状态可能依赖于CaM-KII磷酸化和蛋白磷酸酶去磷酸化之间的平衡，我没有得到支持其他蛋白激酶级联参与反弹增强的结果。据我所知，蛋白激酶A、蛋白激酶C和蛋白激酶G的抑制剂对反跳增强没有显著影响。这些蛋白激酶级联的参与问题将进一步研究使用基因敲除小鼠。少

In cerebellar Purkinje cells, GABAergic inhibitory synaptic transmission undergoes long-lasting "rebound potentiation" following activation of excitatory climbing fiber iputs. Rebound potentiation is triggered by a climbing fiber-induced transient elevation of intracellular Ca^<2+> concentration, and is expressed as a long-lasting increase of postsynaptic GABA_A receptor sensitivity. In the present series of study, I found that inhibitors of the Ca^<2+>/calmodulin-dependent kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. A non-selective protein kinase inhibitor, staurosporine, effectively blocked the induction of rebound potentiation. A calmodulin antagonist, calmidazolium, and a CaM-KII antagonist, KN-62, effectively blocked rebound potentiation, when they were applied extracellularly prior to and during Ca^<2+> channel activation. On the other hand, these antagonists had no effects on the once established rebound potentiation. … More Intracellular application of a peptide antagonist of CaM-KII also blocked the induction of rebound potentiation. These inhibitors had no effects on voltage-gated Ca^<2+> channel currents or on the basal inhibitory transmission itself. Furthermore, intracellular injection of active CaM-KII and application of a phosphatase inhibitor, calyculin A,induced enhancement of GABA_A receptor-mediated currents. These results strongly suggest that RP involves CaM-KII-mediated protein phosphorylation. The functional state of GABA_A receptors may be dependent on the balance between the phosphorylation by CaM-KII and the dephosphorylation by protein phosphatases.I did not obtain the results that support involvement of other protein kinase cascades in rebound potentiation. As far as I examined, inhibitors of protein kinase A,protein kinase C and protein kinase G had no significant effects on rebound potentiation. The issues of involvement of these protein kinase cascades will be further studied using gene knockout mice. Less